Methods for treating cancer using anti-pd-1 antibodies

ABSTRACT

The present invention provides isolated monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to PD-1 with high affinity. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for detecting PD-1, as well as methods for treating various diseases, including cancer and infectious diseases, using anti-PD-1 antibodies. The present invention further provides methods for using a combination immunotherapy, such as the combination of anti-CTLA-4 and anti-PD-1 antibodies, to treat hyperproliferative disease, such as cancer. The invention also provides methods for altering adverse events related to treatment with such antibodies individually.

TECHNICAL FIELD

The present invention relates generally to immunotherapy in thetreatment of human disease and reduction of adverse events relatedthereto. More specifically, the present invention relates to the use ofanti-PD-1 antibodies and the use of combination immunotherapy, includingthe combination of anti-CTLA-4 and anti-PD-1 antibodies, to treat cancerand/or to decrease the incidence or magnitude of adverse events relatedto treatment with such antibodies individually.

BACKGROUND OF THE INVENTION

The protein Programmed Death 1 (PD-1) is an inhibitory member of theCD28 family of receptors, that also includes CD28, CTLA-4, ICOS andBTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells(Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The initialmembers of the family, CD28 and ICOS, were discovered by functionaleffects on augmenting T cell proliferation following the addition ofmonoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansenet al. (1980) Immunogenics 10:247-260). PD-1 was discovered throughscreening for differential expression in apototic cells (Ishida et al.(1992) EMBO J 11:3887-95). The other members of the family, CTLA-4, andBTLA were discovered through screening for differential expression incytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS andCTLA-4 all have an unpaired cysteine residue allowing forhomodimerization. In contrast, PD-1 is suggested to exist as a monomer,lacking the unpaired cysteine residue characteristic in other CD28family members.

The PD-1 gene is a 55 kDa type I transmembrane protein that is part ofthe Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72). PD-1contain a membrane proximal immunoreceptor tyrosine inhibitory motif(ITIM) and a membrane distal tyrosine-based switch motif (ITSM) (Thomas,M. L. (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M (1997)Immunol Today 18:286-91). Although structurally similar to CTLA-4, PD-1lacks the MYPPPY motif that is critical for B7-1 and B7-2 binding. Twoligands for PD-1 have been identified, PD-L1 and PD-L2, that have beenshown to downregulate T cell activation upon binding to PD-1 (Freeman etal. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 andPD-L2 are B7 homologs that bind to PD-1, but do not bind to other CD28family members. One ligand for PD-1, PD-L1 is abundant in a variety ofhuman cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interactionbetween PD-1 and PD-L1 results in a decrease in tumor infiltratinglymphocytes, a decrease in T-cell receptor mediated proliferation, andimmune evasion by the cancerous cells (Dong et al. (2003) J. Mol. Med.81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314;Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppressioncan be reversed by inhibiting the local interaction of PD-1 with PD-L1,and the effect is additive when the interaction of PD-1 with PD-L2 isblocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

PD-1 is an inhibitory member of the CD28 family expressed on activated Bcells, T cells, and myeloid cells (Agata et al., supra; Okazaki et al.(2002) Curr Opin Immunol 14:391779-82; Bennett et al. (2003) J Immunol170:711-8). PD-1 deficient animals develop various autoimmunephenotypes, including autoimmune cardiomyopathy and a lupus-likesyndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity11:141-51; Nishimura et al. (2001) Science 291:319-22). Additionally,PD-1 has been fond to play a role in autoimmune encephalomyelitis,systemic lupus erythematosus, graft-versus-host disease (GVHD), type Idiabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med198:71-78; Prokunina and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143;Nielsen et al. (2004) Lupus 13:510). In a murine B cell tumor line, theITSM of PD-1 was shown to be essential to block BCR-mediated Ca²⁺-fluxand tyrosine phosphorylation of downstream effector molecules (Okazakiet al. (2001) PNAS 98:13866-71).

Accordingly, agents that recognize PD-1 and methods of using suchagents, are desired.

DISCLOSURE OF THE INVENTION

The present invention provides isolated monoclonal antibodies, isparticular human monoclonal antibodies, that bind to PD-1 and thatexhibit numerous desirable properties. These properties include, forexample, high affinity binding to human PD-1, but lacking substantialcross-reactivity with either human CD28, CTLA-4 or ICOS. Still further,antibodies of the invention have been shown to modulate immuneresponses. Accordingly, another aspect of the invention pertains tomethods of modulating immune responses using anti-PD-1 antibodies. Inparticular, the invention provides a method of inhibiting growth oftumor cells in vivo using anti-PD-1 antibodies.

In one aspect, the invention pertains to an isolated monoclonalantibody, or as antigen-binding portion thereof, wherein the antibodyexhibits at least one of the following properties:

(a) binds to human PD-1 with a K_(D) of 1×10⁻⁷ M or less;

(b) does not substantially bind to human CD28, CTLA-4 or ICOS;

(c) increases T-cell proliferation in an Mixed Lymphocyte Reaction (MLR)assay;

(d) Increases interferon-gamma production in an MLR assay;

(e) increases IL-2 secretion in an MLR assay;

(f) binds to human PD-1 and cynomolgus monkey PD-1;

(g) inhibits the binding of PD-L1 and/or PD-L2 to PD-1;

(h) stimulates antigen-specific memory responses;

(i) stimulates antibody responses;

(j) inhibits tumor cell growth in vivo.

Preferably the antibody is a human antibody, although in alternativeembodiments the antibody can be, for example, a murine antibody, achimeric antibody or humanized antibody.

In more preferred embodiments, the antibody binds to human PD-1 with aK_(D) of 5×10⁻⁸ M or less, binds to human PD-1 with a K_(D) of 1×10⁻⁸ Mor less, binds to human PD-1 with a K_(D) of 5×10⁻⁹ M or less, or bindsto human PD-1 with a K_(D) of between 1×10⁻⁸ M and 1×10⁻¹⁰ M.

In another embodiment, the invention provides an isolated monoclonalantibody, or antigen-binding portion thereof, wherein the antibodycross-competes for binding to PD-1 with a reference antibody comprising:

(a) a human heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and7; and

(b) a human light chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13and 14.

In various embodiments, the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 1; and

(h) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 8; or the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 2; and

(h) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 9; or the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 3; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 10; or the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 4; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 11: or the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 5; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 12; or the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 6; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 13; or the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 7; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 14.

In another aspect, the invention pertains to an isolated monoclonalantibody; or an antigen-binding portion thereof, comprising a heavychain variable region that is the product of or derived from a humanV_(H) 3-33 gene, wherein the antibody specifically binds PD-1. Theinvention further provides as isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a heavy chain variableregion that is the product of or derived from a human V_(H) 4-39 gene,wherein the antibody specifically binds PD-1. The invention furtherprovides an isolated monoclonal antibody, or an antigen-binding portionthereof, comprising a light chain variable region that is the product ofor derived from a human V_(K) L6 gene, wherein the antibody specificallybinds PD-1. The invention further provides an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a lightchain variable region that is the product of or derived from a humanV_(K) L15 gene, wherein the antibody specifically binds PD-1.

In a preferred embodiment, the invention provides an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising:

(a) a heavy chain variable region of a human V_(H) 3-33 gene; and

(b) a light chain variable region of a human V_(K) L6 gene;

wherein the antibody specifically binds to PD-1.

In another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising:

(a) a heavy chain variable region of a human V_(H) 4-39 gene; and

(b) a light chain variable region of a human V_(K) L15 gene;

wherein the antibody specifically binds to PD-1.

In another aspect, the invention provides an isolated monoclonalantibody, or antigen-binding portion thereof, comprising:

a heavy chain variable region that comprises CDR1, CDR2, and CDR3sequences; and

a light chain variable region that comprises CDR1, CDR2, and CDR3sequences,

wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 29, 30,31, 32, 33, 34 and 35, and conservative modifications thereof;

(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 50, 51,52, 53, 54, 55 and 56, and conservative modifications thereof; and

(c) the antibody specifically binds to human PD-1.

Preferably, the heavy chain variable region CDR2 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28, and conservativemodifications thereof; and the light chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49, andconservative modifications thereof. Preferably, the heavy chain variableregion CDR1 sequence comprises an amino acid sequence selected from thegroup consisting of amino acid sequences of SEQ ID NOs: 15, 16, 17, 18,19, 20 and 21, and conservative modifications thereof; and the lightchain variable region CDR1 sequence comprises an amino acid sequenceselected from the group consisting of amino acid sequences of SEQ IDNOs: 36, 37, 38, 39, 40, 41 and 42, and conservative modificationsthereof.

In yet another aspect, the invention provides an isolated monoclonalantibody, or antigen-binding portion thereof, comprising a heavy chainvariable region and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14;

(c) the antibody binds to human PD-1 with a K_(D) of 1×10⁻⁷ M or less;and

(d) the antibody does not substantially bind to human CD28, CTLA-4 orICOS.

In a preferred embodiment, the antibodies additionally comprise at leastone of the following properties.

(a) the antibody increases T-cell proliferation in an MLR assay;

(b) the antibody increases interferon-gamma production in an MLR assay;or

(c) the antibody increases IL-2 secretion in an MLR assay.

Additionally or alternatively, the antibody may comprise one or more ofthe other features listed above.

In preferred embodiments, the invention provides an isolated monoclonalantibody, or antigen-binding portion thereof, comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20and 21;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27and 28;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34and 35;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41and 42;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48and 49; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55and 56;

wherein the antibody specifically binds PD-1.

A preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 22;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 29;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 36;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 43; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 50.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 16;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 23;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 30;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 37;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 44; and

(f ) a light chain variable region CDR3 comprising SEQ ID NO: 51.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 17;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 31;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 38;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 45; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 52.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 18;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 25;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 32;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 39;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 46; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 53.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 19;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 26;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 33;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 40;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 47; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 54.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 20;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 27;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 34;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 41;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 48; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 55.

Another preferred combination comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 21;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 28;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 35;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 42;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 49; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 56.

Other preferred antibodies of the invention, or antigen-binding portionsthereof, comprise:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and7; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13and 14;

wherein the antibody specifically binds PD-1.

A preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 1; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 8.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 2; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 9.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 3; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 10.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 4; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 11.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 5; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 12.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 6; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 13.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 1; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 14.

The antibodies of the invention can be, for example, full-lengthantibodies, for example of an IgG1 or IgG4 isotype. Alternatively, theantibodies can be antibody fragments, such as Fab or Fab′2 fragments, orsingle chain antibodies.

The invention also provides an immunoconjugate comprising an antibody ofthe invention, or antigen-binding portion thereof, linked to atherapeutic agent, such as a cytotoxin or a radioactive isotope. Theinvention also provides a bispecific molecule comprising an antibody, orantigen-binding portion thereof, of the invention, linked to a secondfunctional moiety having a different binding specificity than saidantibody, or antigen-binding portion thereof.

Compositions comprising an antibody, or antigen-binding portion thereof,or immunoconjugate or bispecific molecule of the invention, and apharmaceutically acceptable carrier, are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of the invention are also encompassed by theinvention, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. Moreover, theinvention provides a transgenic mouse comprising human immunoglobulinheavy and light chain transgenes, wherein the mouse expresses anantibody of the invention, as well as hybridomas prepared from such amouse, wherein the hybridoma produces the antibody of the invention.

In yet another aspect, the invention provides a method of modulating asimmune response in a subject comprising administering to the subject theantibody, or antigen-binding portion thereof, of the invention such thatthe immune response in the subject is modulated. Preferably, theantibody of the invention enhances, stimulates or increases the immuneresponse in the subject.

In a further aspect, the invention provides a method of inhibitinggrowth of tumor cells in a subject, comprising administering to asubject a therapeutically effective amount of an anti-PD-1 antibody, orantigen-binding portion thereof. The antibodies of the invention arepreferred for use in the method although other anti-PD-1 antibodies canbe used instead (or in combination with an anti-PD-1 antibody of theinvention). For example, a chimeric, humanized or fully human anti-PD-1antibody can be used in the method of inhibiting tumor growth.

In a further aspect, the invention provides a method of treating aninfections disease in a subject, comprising administering to a subject atherapeutically effective amount of an anti-PD-1 antibody, orantigen-binding portion thereof. The antibodies of the invention arepreferred for use in the method although other anti-PD-1 antibodies canbe used instead (or in combination with an anti-PD-1 antibody of theinvention). For example, a chimeric, humanized or fully human anti-PD-1antibody can be used in the method of treating an infectious disease.

Still further, the invention provides a method of enhancing an immuneresponse to an antigen in a subject comprising administering to thesubject: (i) the antigen; and (ii) an anti-PD-1 antibody, orantigen-binding portion thereof, such that an immune response to theantigen in the subject is enhanced. The antigen can be, for example, atumor antigen, a viral antigen, a bacterial antigen or an antigen from apathogen. The antibodies of the invention are preferred for use in themethod although other anti-PD-1 antibodies can be used instead (or incombination with an anti-PD-1 antibody of the invention). For example, achimeric, humanized or fully human anti-PD-1 antibody can be used in themethod of enhancing an immune response to an antigen in a subject.

The invention also provides methods for making “second generation”anti-PD-1 antibodies based on the sequences of the anti-PD-1 antibodiesprovided herein. For example, the invention provides a method forpreparing an anti-PD-1 antibody comprising:

-   -   (a) providing: (i) a heavy chain variable region antibody        sequence comprising a CDR1 sequence that is selected from the        group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21,        and/or a CDR2 sequence that is selected from the group        consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28; and/or        a CDR3 sequence that is selected from the group consisting of        SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35; or (ii) a light chain        variable region antibody sequence comprising a CDR1 sequence        that is selected from the group consisting of SEQ ID NOs: 36,        37, 38, 39, 40, 41 and 42, and/or a CDR2 sequence that is        selected from the group consisting of SEQ ID NOs: 43, 44, 45,        46, 47, 41 and 49, and/or a CDR3 sequence that is selected from        the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and        56;    -   (b) altering at least one amino acid residue within at least one        variable region antibody sequence, said sequence being selected        from the heavy chain variable region antibody sequence and the        light chain variable region antibody sequence, to create at        least one altered antibody sequence; and    -   (c) expressing the altered antibody sequence as a protein.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting. The contents of all references, GenBank entries,patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 57) and amino acidsequence (SEQ ID NO: 1) of the heavy chain variable region of the 17D8human monoclonal antibody. The CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO:22) and CDR3 (SEQ ID NO: 29) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 64) and amino acidsequence (SEQ ID NO: 8) of the light chain variable region of the 17D8human monoclonal antibody. The CDR1 (SEQ ID NO: 36), CDR2 (SEQ ID NO:43) and CDR3 (SEQ ID NO: 50) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 58) and amino acidsequence (SEQ ID NO: 2) of the heavy chain variable region of the 2D3human monoclonal antibody. The CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO:23) and CDR3 (SEQ ID NO: 30) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 65) and amino acidsequence (SEQ ID NO: 9) of the light chain variable region of the 2D3human monoclonal antibody. The CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO:44) and CDR3 (SEQ ID NO: 51) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 59) and amino acidsequence (SEQ ID NO: 3) of the heavy chain variable region of the 4H1human monoclonal antibody. The CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO:24) and CDR3 (SEQ ID NO: 31) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 66) and amino acidsequence (SEQ ID NO: 10) of the light chain variable region of the 4H1human monoclonal antibody. The CDR1 (SEQ ID NO: 38), CDR2 (SEQ ID NO:45) and CDR3 (SEQ ID NO: 52) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 60) and amino acidsequence (SEQ ID NO: 4) of the heavy chain variable region of the 5C4human monoclonal antibody. The CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO:25) and CDR3 (SEQ ID NO: 32) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 67) and amino acidsequence (SEQ ID NO: 11) of the light chain variable region of the 5C4human monoclonal antibody. The CDR1 (SEQ ID NO: 39), CDR2 (SEQ ID NO:46) and CDR3 (SEQ ID NO: 53) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 61) and amino acidsequence (SEQ ID NO: 5) of the heavy chain variable region of the 4A11human monoclonal antibody. The CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO:26) and CDR3 (SEQ ID NO: 33) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 68) and amino acidsequence (SEQ ID NO: 12) of the light chain variable region of the 4A11human monoclonal antibody. The CDR1 (SEQ ID NO: 40), CDR2 (SEQ ID NO:47) and CDR3 (SEQ ID NO: 54) regions are delineated and the Y and Jgermline derivations are indicated.

FIG. 6A shows the nucleotide sequence (SEQ ID NO: 62) and amino acidsequence (SEQ ID NO: 6) of the heavy chain variable region of the 7D3human monoclonal antibody. The CDR1 (SEQ ID NO: 20), CDR2 (SEQ ID NO:27) and CDR3 (SEQ ID NO: 34) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO: 69) and amino acidsequence (SEQ ID NO: 13) of the light chain variable region of the 7D3human monoclonal antibody. The CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO:48) and CDR3 (SEQ ID NO: 55) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 7A shows the nucleotide sequence (SEQ ID NO: 63) and amino acidsequence (SEQ ID NO: 7) of the heavy chain variable region of the 5F4human monoclonal antibody. The CDR1 (SEQ ID NO: 21), CDR2 (SEQ ID NO:28) and CDR3 (SEQ ID NO: 35) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 7B shows the nucleotide sequence (SEQ ID NO: 70) and amino acidsequence (SEQ ID NO: 14) of the light chain variable region of the 5F4human monoclonal antibody. The CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO:49) and CDR3 (SEQ ID NO: 56) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 8 shows the alignment of the amino acid sequence of the heavy chainvariable region of 17D8, 2D3, 4H1, 5C4 and 7D3 with the human germlineV_(H) 3-33 amino acid sequence (SEQ ID NO: 71).

FIG. 9 shows the alignment of the amino acid sequence of the light chainvariable region of 17D8, 2D3 and 7D3 with the human germline V_(k) L6amino acid sequence (SEQ ID NO: 73).

FIG. 10 shows the alignment of the amino acid sequence of the lightchain variable region of 4H1 and 5C4 with the human germline V_(k) L6amino acid sequence (SEQ ID NO: 73).

FIG. 11 shows the alignment of the amino acid sequence of the heavychain variable region of 4A11 and 5F4 with the human germline V_(H) 4-39amino acid sequence (SEQ ID NO: 72).

FIG. 12 shows the alignment of the amino acid sequence of the lightchain variable region of 4A11 and 5F4 with the human germline V_(k) L15amino acid sequence (SEQ ID NO: 74).

FIGS. 13A-13B show the results of flow cytometry experimentsdemonstrating that the human monoclonal antibodies 5C4 and 4H1, directedagainst human PD-1, binds the cell surface of CHO cells transfected withfull-length human PD-1. FIG. 13A shows to the flow cytometry plot for5C4. FIG. 13B shows the flow cytometry plot for 4H1. Thin linerepresents the binding to CHO cells and solid line represents thebinding to CHO hPD-1 cells.

FIG. 14 shows a graph demonstrating that the human monoclonal antibodies17D8, 2D3, 4H1, 5C4, and 4A11, directed against human PD-1, bindspecifically to PD-1, and not to other members of the CD28 family.

FIGS. 15A-15C show the results of flow cytometry experimentsdemonstrating that the human monoclonal antibodies 4H1 and 5C4, directedagainst human PD-1, binds to PD-1 on the cell surface. FIG. 15A showsbinding to activated human T-cells. FIG. 15B shows the binding tocynomolgous monkey T-cells. FIG. 15C shows the binding to CHOtransfected cells expressing PD-1.

FIGS. 16A-16C show the results of experiments demonstrating that humanmonoclonal antibodies against human PD-1 promote T-cell proliferation.IFN-gamma secretion and IL-2 secretion in a mixed lymphocyte reactionassay. FIG. 16A is a bar graph showing concentration dependent T-cellproliferation; FIG. 16B is a bar graph showing concentration dependentIFN-gamma secretion; FIG. 16C is a bar graph showing concentrationdependent IL-2 secretion.

FIGS. 17A-17B show the results of flow cytometry experimentsdemonstrating that human monoclonal antibodies against human PD-1 blockthe binding of PD-L1 and PD-L2 to CHO transfected cells expressing PD-1.FIG. 17A is a graph showing inhibition of binding of PD-L1; FIG. 17B isa graph showing inhibition of binding of PD-L2.

FIG. 18 shows the results of flow cytometry experiments demonstratingthat human monoclonal antibodies against human PD-1 do not promoteT-cell apoptosis.

FIG. 19 shows the results of experiments demonstrating that anti-PD-1HuMabs have a concentration dependent effect on IFN gamma secretion byPBMCs from CMV-positive donors when PBMCs were stimulated with a CMVlysate and anti-PD-1.

FIG. 20 shows the results of tumor growth experiments in a mouse modelsystem demonstrating that treatment in vivo of mouse tumors withanti-PD-1 antibodies inhibits the growth of tumors.

FIGS. 21A to 21D show the tumor volume over time in individual mice thatwere implanted with colon tumor cells (PD-L1⁻) and on the same daytreated with one of the following merges: (A) mouse IgG (control), (B)anti-CTLA-4 antibody, (C) anti-PD-1 antibody, and (D) anti-CTLA-4antibody and anti-PD-1 antibody. The mice received subsequent antibodytreatments on days 3, 6 and 10 as described in Example 13 and tumorvolume was monitored over 60 days.

FIG. 22 shows the mean tumor volume of the mice shown in FIG. 21.

FIG. 23 shows the median tumor volume of the mice shown in FIG. 21.

FIGS. 24A to 24D show the tumor volume over time in individual mice thatwere implanted with MC38 colon tumor cells (PD-L1⁻) and one week latertreated with one of the following therapies: (A) mouse IgG (control),(B) anti-CTLA-4 antibody, (C) anti-PD-1 antibody, and (D) anti-CTLA-4antibody and anti-PD-1 antibody. The tumor volume on the first day oftreatment was about 315 mm³. The mice received subsequent antibodytreatments on days 3, 6 and 10 as described in Example 14.

FIG. 25 shows the mean tumor volume of the mice shown in FIG. 24.

FIG. 26 shows the median tumor volume of the mice shown in FIG. 24,

FIG. 27 shows the mean tumor volume over time in individual mice thatwere implanted with MC38 colon tumor cells (PD-L1⁻) (day −7) and thentreated on days 0, 3, 6 and 10 post-implantation (as described inExample 15) with one of the following therapies: (A) mouse IgG as acontrol (20 mg/kg, X₂₀) (B) anti-PD-1 antibody (10 mg/kg) and mouse IgG(10 mg/kg) (P₁₀X₁₀), (C) anti-CTLA-4 antibody (10 mg/kg) and mouse IgG(10 mg/kg) (C₁₀X₁₀), (D) anti-CTLA-4 antibody and anti-PD-1 antibody (10mg/kg each) (C₁₀P₁₀), (E) anti-CTLA-4 antibody and anti-PD-1 antibody (3mg/kg each) (C₃P₃), and (F) anti-CTLA-4 antibody and anti-PD-1 antibody(1 mg/kg each) (C₁P₁). Two groups of mice were treated with eachantibody sequentially as follows: (G) anti-CTLA-4 antibody (10 mg/kg,day 0), anti-CTLA-4 antibody (10 mg/kg, day 3), anti-PD-1 antibody (10mg/kg, day 6), and anti-PD-1 antibody (10 mg/kg, day 10) (C₁₀C₁₀P₁₀P₁₀);and (H) anti-PD-1 antibody (10 mg/kg, day 0), anti-PD-1 antibody (10mg/kg, day 3), anti-CTLA-4 antibody (10 mg/kg, day 6), and anti-CTLA-4antibody (10 mg/kg, day 10) (10 mg/kg, day 10) (P₁₀P₁₀C₁₀C₁₀).

FIG. 28 shows the mean tumor volume of the mice shown in FIG. 27.

FIG. 29 shows the median tumor volume of the mice shown in FIG. 27.

FIGS. 30A to 30F show the tumor volume over time in individual mice thatwere implanted with SA1/N fibrosarcoma cells (PD-L1⁻) and one day latertreated with one of the following therapies: (A) PBS (vehicle control),(B) mouse IgG (antibody control, 10 mg/kg), (C) anti-PD-1 antibody (10mg/kg), (D) anti-CTLA-4 antibody (10 mg/kg), (E) anti-CTLA-4 antibody(0.2 mg/kg), and (F) anti-PD-1 antibody (10 mg/kg) and anti-CTLA-4antibody (0.2 mg/kg). The mice received subsequent antibody treatmentson days 4, 7 and 11 as described in Example 16 and tumor volume wasmonitored over 41 days.

FIG. 31 shows the mean tumor volume of the mice shown in FIG. 29.

FIG. 32 shows the median tumor volume of the mice shown in FIG. 29.

FIGS. 33A to 33J show the tumor volume over time in individual mice thatwere implanted with SA1/N fibrosarcoma cells (PD-L1⁻) and then treatedon days 7, 10, 13 and 17 post-implantation (as described in Example 17)with one of the following therapies: (A) PBS (vehicle control), (B)mouse IgG (antibody control, 10 mg/kg), (C) anti-CTLA-4 antibody (0.25mg/kg), (D) anti-CTLA-4 antibody (0.5 mg/kg), (E) anti-CTLA-4 antibody(5 mg/kg), (F) anti-PD-1 antibody (3 mg/kg), (G) anti-PD-1 antibody (10mg/kg), (H) anti-PD-1 antibody (10 mg/kg) and anti-CTLA-4 antibody (0.25mg/kg), (I) anti-PD-1 antibody (10 mg/kg) and anti-CTLA-4 antibody (0.5mg/kg), and (F) anti-PD-1 antibody (3 mg/kg) and anti-CTLA-4 antibody(0.5 mg/kg). The tumor volume on the first day of treatment was about110 mm³.

FIG. 34 shows the mean tumor volume of the mice shown in FIG. 33.

FIG. 35 shows the median tumor volume of the mice shown in FIG. 33.

FIGS. 36A and 36B show the tumor volume over time in individual micethat were implanted with SA1/N fibrosarcoma cells (PD-L1⁻) and thentreated on days 10, 13, 16 and 19 post-implantation (as described inExample 17) with one of the following therapies: (A) mouse IgG (antibodycontrol, 10 mg/kg) or (B) anti-PD-1 antibody (1.0 mg/kg) andanti-CTLA-4antibody (1 mg/kg). The tumor volume on the first day oftreatment was about 250 mm³.

FIG. 37 shows the mean tumor volume of the mice shown in FIG. 36.

FIG. 38 shows the median tumor volume of the mice shown in FIG. 36.

FIG. 39 shows the mean and median percent tumor inhibition calculatedfrom the tumor volumes shown in FIGS. 33 and 36.

FIGS. 40A to 40D show the tumor volume in BALB/c mice that wereimplanted subcutaneously with RENCA renal adenocarcinoma cells (PD-L1⁺)(Murphy and Hrushesky (1973), J. Nat'l. Cancer Res. 50:1013-1025) (day−12) and then treated intraperitoneally on days 0, 3, 6 and 9post-implantation with one of the following therapies: (A) mouse IgG(antibody control 20 mg/kg), (B) anti-PD-1 antibody (10 mg/kg), (C)anti-CTLA-4 antibody (10 mg/kg), and (D) anti-PD-1 antibody (10 mg/kg)in combination with anti-CTLA-4 antibody (10 mg/kg). The tumor volume onthe first day of treatment was about 115 mm³.

FIG. 41 shows binding of mouse PD-L2-Fc fusion protein to mouse PD-1(mPD-1) is blocked by anti-mPD-1 antibody 4H2 in a dose dependentmanner. The binding is detected by measuring fluorescence ofFITC-labeled donkey-anti-rat IgG by ELISA. The greater the MFI (meanfluorescence intensity) the greater the binding.

FIG. 42 shows binding curves of anti-mPD-1 antibodies to immobilizedmPD-1-Fc fusion protein by ELISA.

FIG. 43 shows the binding curve of rat anti-mPD-1 antibody 4H2, B3 tomPD-1-expressing CHO cells. Binding was detected with donkey-anti-ratIgG, FITC conjugated and measured by FACS (MFI).

FIG. 44 shows the binding curve of mPD-L1-hFc fusion protein tomPD-1-expressing CHO cells in the presence of increasing concentrationsof anti-mPD-1 antibody 4H2.B3. Binding was detected with goat-anti-humanIgG, FITC conjugated and measured by FACS (MFI).

FIG. 45 shows the binding curves of rat anti-mPD-1 antibody 4H2.B3 tomPD-1-expressing CHO cells as compared to chimeric rat:mouse anti-mPD-1antibody 4H2.

FIG. 46 shows the binding curves of mPD-L1-hFc fusion protein tomPD-1-expressing CHO cells in the presence of increasing concentrates ofeither rat anti-mPD-1 antibody 4H2.B3 or chimeric rat:mouse anti-mPD-1antibody 4H2.

FIG. 47 shows the mean tumor volume of tumor-free mice previouslytreated with anti-PD1 antibody and re-challenged with SA1/N fibrosarcomacells (PD-L1⁻). Also shown is the mean tumor volume of naïve mice(control, not previously challenged, or treated) implanted with SA1/Nfibrosarcoma cells.

FIG. 48 shows the tumor volume over time in individual mice, whichsurvived tumor-free following implantation of MC38 colon tumor cells(PD-L1⁻) and treatment with anti-PD1 antibody or a combination ofanti-PD1 antibody with anti-CTLA-4 antibody), re-challenged with 10×moreMC38 colon tumor cells than the initial treatment. Also shown is themean tumor volume of naïve mice (control, not previously challenged ortreated) implanted with MC38 colon tumor cells.

FIG. 49 shows the mean tumor volume of the mice shown in FIG. 48.

FIG. 50 shows the mean tumor volume over time in individual mice thatwere implanted with CT26 colon tumor cells.

FIGS. 51A-B shows the results of experiments demonstrating that humanmonoclonal antibodies against human PD-1 promote T-cell proliferationand IFN-gamma secretion in cultures containing T regulatory cells. FIG.50A is a bar graph showing concentration dependent T-cell proliferationusing HuMAb 5C4; FIG. 50B is a bar graph showing concentration dependentIFN-gamma secretion using HuMAb 5C4.

FIGS. 52A-B shows the results of experiments demonstrating that humanmonoclonal antibodies against human PD-1 promote T-cell proliferationand IFN-gamma secretion in cultures containing activated T cells. FIG.51A is a bar graph showing concentration dependent T-cell proliferationusing HuMAb 5C4; FIG. 51B is a bar graph showing concentration dependentIFN-gamma secretion using HuMAb 5C4.

FIG. 53 shows the results of an antibody dependent cellular cytotoxicity(ADCC) assay demonstrating that human monoclonal anti-PD-1 antibodieskill human activated T cells in an ADCC concentration-dependent mannerin relation to the Fc region of the anti-PD-1 antibody.

FIG. 54 shows the results of a complement dependent cytotoxicity (CDC)assay demonstrating that human monoclonal anti-PD-1 antibodies do notkill human activated T cells in a CDC concentration-dependent manner.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to isolated monoclonalantibodies, particularly human monoclonal antibodies, that bindspecifically to PD-1. In certain embodiments, the antibodies of theinvention exhibit one or more desirable functional properties, such ashigh affinity binding to PD-1, lack of cross-reactivity to other CD28family members, the ability to stimulate T cell proliferation, IFN-γand/or IL-2 secretion in mixed lymphocyte reactions, the ability toinhibit binding of one or more PD-1 ligands (e.g., PD-L1 and/or PD-L2),the ability to cross-react with cynomolgus monkey PD-1, the ability tostimulate antigen-specific memory responses, the ability to stimulateantibody responses and/or the ability to inhibit growth of tumor cellsin vivo. Additionally or alternatively, the antibodies of the inventionare derived from particular heavy and light chain germline sequencesand/or comprise particular structural features such as CDR regionscomprising particular amino acid sequences. In another aspect, theinvention relates to the combined use of monoclonal antibodies that bindspecifically to PD-1 and monoclonal antibodies that bind specifically toCTLA-4.

The invention provides, for example, isolated antibodies, methods ofmaking such antibodies, immunconjugates and bispecific moleculescomprising such antibodies and pharmaceutical compositions containingthe antibodies, immunoconjugates or bispecific molecules of theinvention.

In another aspect, the invention pertains to methods of inhibitinggrowth of tumor cells in a subject using anti-PD-1 antibodies. Asdemonstrated herein, anti-PD-1 antibodies are capable of inhibitingtumor cell growth in vivo. The invention also relates to methods ofusing the antibodies to modify an immune response, as well as to treatdiseases such as cancer or infectious disease, or to stimulate aprotective autoimmune response or to stimulate antigen-specific immuneresponses (e.g., by coadministration of anti-PD-1 with an antigen ofinterest).

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “Programmed Death 1,” “Programmed Cell Death 1,” “ProteinPD-1,” “PD-1,” “PD1,” “PDCD1,” “hPD-1” and “hPD-1” are usedinterchangeably, and include variants, isoforms, species homologs ofhuman PD-1, and analogs having at least one common epitope with PD-1.The complete PD-1 sequence can be found under GenBank Accession No.U64863.

The terms “cytotoxic T lymphocyte-associated antigen-4,” “CTLA-4,”“CTLA4,” “CTLA-4 antigen” and “CD152” (see, e.g., Murata, Am. J. Pathol.(1999) 115:453-450) are used interchangeably, and include variants,isoforms, species homologs of human CTLA-4, and analogs having at leastone common epitope with CTLA-4 (see, e.g., Balzano (1992) Int. J. CancerSuppl. 7:28-32). The complete CTLA-4 nucleic acid sequence can be foundunder GenBank Accession No. L15006.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present invention is the PD-1 receptor.

The term “antibody” as referred to herein includes whole antibodies andany antigen-binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen-binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., PD-1). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(l) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) as isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA b 85:5879 -5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds PD-1 is substantially free of antibodies that specifically bindantigens other than PD-1). An isolated antibody that specifically bindsPD-1 may, however, have cross-reactivity to other antigens, such as PD-1molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived front another species, such as anantibody in winch the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binds to human PD-1” isintended to refer to an antibody that binds to human PD-1 with a K_(D)of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less, more preferably1×10⁻⁸ M or less, more preferably 5×10⁻⁹ M or less.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a K_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M orless, even more preferably 10⁻⁹ M or less.

The term “treatment” or “therapy” refers to administering an activeagent with the purpose to core, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect a condition (e.g., a disease), thesymptoms of the condition, or to prevent or delay the onset of thesymptoms, complications, biochemical indicia of a disease, or otherwisearrest or inhibit further development of the disease, condition, ordisorder in a statistically significant manner.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended, even undesirable, sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. For example, an adverse event may be associated withactivation of the immune system or expansion of immune system, cells(e.g., T cells) in response to a treatment. A medical treatment may haveone or more associated AEs and each AE may have the same or differentlevel of severity. Reference to methods capable of “altering adverseevents” means a treatment regime that decreases the incidence and/orseverity of one or more AEs associated with the use of a differenttreatment regime.

As used herein, “hyperproliferative disease” refers to conditionswherein cell growth is increased over normal levels. For example,hyperproliferative diseases or disorders include malignant diseases(e.g., esophageal cancer, colon cancer, biliary cancer) andnon-malignant diseases (e.g., atherosclerosis, benign hyperplasia,benign prostatic hypertrophy).

As used herein, “subtherapeutic dose” means a dose of a therapeuticcompound (e.g., an antibody) that is lower than the usual or typicaldose of the therapeutic compound when administered alone for thetreatment of a hyperproliferative disease (e.g., cancer). For example, asubtherapeutic dose of CTLA-4 antibody is a single dose of the antibodyat less than about 3 mg/kg, i.e., the known dose of anti-CTLA-4antibody.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component

As used herein, “about” or “comprising essentially of” mean within anacceptable error range tor the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 20%. Furthermore, particularly with respect to biologicalsystems or processes, the terms can mean up to an order of magnitude orup to 5-fold of a value. When particular values are provided in theapplication and claims, unless otherwise stated, the meaning of “about”or “comprising essentially of” should be assumed to be within anacceptable error range for that particular value.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

As used herein, the term “subject” includes any human ornonhuman-animal. The term “nonhuman animal” includes all vertebrates,e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs,cats, horses, cows chickens, amphibians, reptiles, etc. Except whennoted, the terms “patient” or “subject” are used interchangeably.

Various aspects of the invention are described is further detail in thefollowing subsections.

Anti-PD-1 Antibodies

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind specifically to PD-1 (e.g., bind to human PD-1 and maycross-react with PD-1 from other species, such as cynomolgus monkey).Preferably, an antibody of the invention binds to PD-1 with highaffinity, for example with a K_(D) of 1×10⁻⁷ M or less. The anti-PD-1antibodies of the invention preferably exhibit one or more of thefollowing characteristics:

(a) binds to human PD-1 with a K_(D) of 1×10⁻⁷ M or less;

(b) does not substantially bind to human CD28, CTLA-4 or ICOS;

(c) increases T-cell proliferation in an Mixed Lymphocyte Reaction (MLR)assay;

(d) increases interferon-gamma production in an MLR assay;

(e) increases IL-2 secretion in an MLR assay;

(f) binds to human PD-1 and cynomolgus monkey PD-1;

(g) inhibits the binding of PD-L1 and/or PD-L2 to PD-1;

(h) stimulates antigen-specific memory responses;

(i) stimulates antibody responses;

(j) inhibits tumor cell growth in vivo.

Preferably, the antibody binds to human PD-1 with a K_(D) of 5×10⁻⁸ M orless, binds to human PD-1 with a K_(D) of 1×10⁻⁸ M or less, binds tohuman PD-1 with a K_(D) of 5×10⁻⁹ M or less, or binds to human PD-1 witha K_(D) of between 1×10⁻⁸ M and 1×10⁻⁰¹ M or less.

An antibody of the invention may exhibit any combination of theabove-listed features, such as two, three, four, five or more of theabove-listed features.

Standard assays to evaluate the binding ability of the antibodies towardPD-1 are known in the art, including for example, ELISAs, Western blotsand RIAs. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by Biacore analysis. Suitable assays for evaluating any of theabove-described characteristics are described in detail in the Examples.

Monoclonal Antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4

Preferred antibodies of the invention are the human monoclonalantibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 3F4 isolated andstructurally characterized as described in Examples 1 and 2. The V_(H)amino acid sequences of 17D8, 2D3, 4 H1, 5C4, 4A11, 7D3 and 5F4 areshown in SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7, respectively. The V_(L)amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shownin SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14, respectively.

Given that each of these antibodies can bind to PD-1, the V_(H) andV_(L) sequences can be “mixed and matched” to create other anti-PD-1binding molecules of the invention. PD-1 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISAs). Preferably, when V_(H) andV_(L) chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence.

Accordingly, in one aspect, the invention provides an isolatedmonoclonal antibody, or antigen-binding portion thereof comprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and7; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13and 14;

wherein the antibody specifically binds PD-1, , preferably human PD-1.

Preferred heavy and light chain combinations include:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 1; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 8; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 2; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 9; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 3; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 10; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 4; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 11; or

(a) a heavy chain, variable region comprising the amino acid sequence ofSEQ ID NO: 5; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 12; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 6; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 13; or

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 7; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 14.

In another aspect, the invention provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of 17D8, 2D3, 4H1,5C4, 4A11, 7D3 and 5P4, or combinations thereof. The amino acidsequences of the V_(H) CDR1s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4are shown in SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21, respectively.The amino acid sequences of the V_(H) CDR2s of 17D8, 2D3, 4H1, 5C4,4A11, 7D3, and 5F4 are shown in SEQ ID NOs: 22, 23, 24, 25, 26, 27 and28, respectively. The amino acid sequences of the V_(H) CDR3s of 17D8,2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 29, 30, 31,32, 33, 34 and 35, respectively. The amino acid sequences of the V_(k)CDR1s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs:36, 37, 38, 30, 40, 41 and 42, respectively. The amino acid sequences ofthe V_(k) CDR2s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown inSEQ ID NOs: 43, 44, 45, 46, 47, 47, 48 and 49, respectively. The aminoacid sequences of the V_(k) CDR3s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and5F4 are shown in SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56,respectively. The CDR regions are delineated using the Kabat system(Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242).

Given that each of these antibodies can bind to PD-1 and thatantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(k) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a V_(H) CDR1, CDR2, and CDR3 and a V_(k) CDR1, CDR2, and CDR3)to create other anti-PD-1 binding molecules of the invention. PD-1binding of such “mixed and matched” antibodies can be tested using thebinding assays described above and in the Examples (e.g., ELISAs,Biacore analysis). Preferably, when V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(k) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(k) sequence preferably isreplaced with a structurally similar CDR sequence(s). It will be readilyapparent to the ordinarily skilled artisan that novel V_(H) and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies antibodies 17D8,2D3, 4H1, 5C4, 4A11, 7D3 and 5F4.

Accordingly, in another aspect, the invention provides an isolatedmonoclonal antibody, or antigen-binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20and 21;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27and 28;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34and 35;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41and 42;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48and 49; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55and 56;

wherein the antibody specifically binds PD-1, preferably human PD-1.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 22;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 29;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 36,

(e) a light chain variable region CDR2 comprising SEQ ID NO: 43; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 50.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 16;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 23;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO. 30;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 37;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 44; and

(f) a light chain variable region CDR1 comprising SEQ ID NO: 51.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 17;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24,

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 31;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 38;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 45; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 52.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 18;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 25,

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 32;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 39;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 46; and

(f) a fight chain variable region CDR3 comprising SEQ ID NO: 53.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 19;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 26;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 33;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 40;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 47; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 54.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of the invention comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the invention provides anisolated monoclonal antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 3-33 gene, wherein the antibody specificallybinds PD-1, preferably human PD-1. In another preferred embodiment, theinvention provides an isolated monoclonal antibody, of anantigen-binding portion thereof, comprising a heavy chain variableregion that is the product of or derived from a human V_(H) 4-39 gene,wherein the antibody specifically binds PD-1, preferably human PD-1. Inyet another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(K) L6 gene, wherein the antibody specifically binds PD-1,preferably human PD-1. In yet another preferred embodiment, theinvention provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a light chain variableregion that is the product of or derived from a human V_(K) L15 gene,wherein the antibody specifically binds PD-1, preferably human PD-1. Inyet another preferred embodiment, the invention provides an isolatedmonoclonal antibody, or antigen-binding portion thereof, wherein theantibody:

(a) composes a heavy chain variable region that is the product of orderived from a human V_(H) 3-33 or 4-39 gene (which gene encodes theamino acid sequence set forth in SEQ ID NO: 71 or 73, respectively);

(b) comprises a light chain variable region that is the product of orderived from a human V_(K) L6 or L15 gene (which gene encodes the aminoacid sequence set forth in SEQ ID NO: 72 or 74, respectively); and

(c) specifically binds to PD-1.

Examples of antibodies having V_(H) and V_(K) of V_(H) 3-33 and V_(K)L6, respectively, are 17D8, 2D3, 4H1, 5C4, and 7D3. Examples ofantibodies having V_(H) and V_(K) of V_(H) 4-39 and V_(K) L15,respectively are 4A11 and 5F4.

As used herein, a human antibody comprises heavy, or light chainvariable regions that is “the product of” or “derived from” a particulargermline sequence if the variable regions of the antibody are obtainedfrom a system that uses human germline immunoglobulin genes. Suchsystems include immunizing a transgenic mouse carrying humanimmunoglobulin genes with the antigen of interest or screening a humanimmunoglobulin gene library displayed on phage with the antigen ofinterest. A human antibody that is “the product of” or “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequences of human germline immunoglobulins and selecting the humangermline immunoglobulin sequence that is closest in sequence (i.e.,greatest % identity) to the sequence of the human antibody. A humanantibody that is “the product of” or “derived from” a particular humangermline immunoglobulin sequence may contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a selected human antibody typically isat least 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human antibody as being human when comparedto the germline immunoglobulin amino acid sequences of other species(e.g., murine germline sequences). In certain cases, a human antibodymay be at least 95%, or even at least 96%, 97%, 98%, or 99% identical inamino acid sequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention comprises heavyand light chain variable regions comprising amino acid that arehomologous to the amino acid sequences of the preferred-antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties of the anti-PD-1 antibodies of the invention.

For example, the invention provides an isolated monoclonal antibody, orantigen-binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 8, 9, 10, 11, 11, 12, 13 and 14; and

the antibody exhibits one or more of the following properties:

(c) the antibody binds to human PD-1 with a K_(D) of 1×10⁻⁷ M or less;

(d) the antibody does not substantially bind to human CD28, CTLA-4 orICOS;

(e) the antibody increases T-cell proliferation in an MLR assay;

(f) the antibody increases interferon-gamma production in an MLR assay;

(g) the antibody increases Il-2 secretion in an MLR assay;

(h) the antibody binds to human PD-1 and cynomolgus monkey PD-1;

(i) the antibody inhibits the binding of PD-L1 and/or PD-L2 to PD-1;

(j) the antibody stimulates antigen-specific memory responses;

(k) the antibody stimulates antibody responses;

(l) the antibody inhibits tumor cell growth in vivo.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An anybody having V_(H) and V_(L) regions having high(i.e., 80% or greater) homology to the V_(H) and V_(L) regions of thesequences set forth above, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs: 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69and 70, followed by testing of the encoded altered antibody for retainedfunction (i.e., the functions set forth in (c) through (l) above) usingthe functional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g.,17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4), or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the anti-PD-1 antibodies of the invention. Accordingly,the invention provides an isolated monoclonal antibody, orantigen-binding portion thereof, comprising a heavy chain variableregion comprising CDR1, CDR2, and CDR3 sequences and a light chainvariable region comprising CDR1, CDR2, and CDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35, and conservativemodifications thereof;

(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequenceof SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56, and conservativemodifications thereof; and

the antibody exhibits one or more of the following properties:

(c) the antibody binds to human PD-1 with a K_(D) of 1×10⁻⁷ M or leas;

(d) the antibody does not substantially bind to human CD28, CTLA-4 orICOS;

(e) the antibody increases T-cell proliferation in an MLR assay;

(f) the antibody increases interferon-gamma production in an MLR assay;

(g) the antibody increases Il-2 secretion in an MLR assay;

(h) the antibody binds to human PD-1 and cynomolgus monkey PD-1;

(i) the antibody inhibits the binding of PD-L1 and/or PD-L2 to PD-1;

(j) the antibody stimulates antigen-specific memory responses;

(k) the antibody stimulates antibody responses;

(l) the antibody inhibits tumor cell growth in vivo.

In a preferred embodiment, the heavy chain variable region CDR3 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28, andconservative modifications thereof; and the light chain variable regionCDR2 sequence comprises an amino acid sequence selected from the groupconsisting of amino acid sequences of SEQ ID NOs: 43, 44, 45, 46, 47, 48and 49, and conservative modifications thereof. In another preferredembodiment, the heavy chain variable region CDR1 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and sad 21, andconservative modifications thereof; and the light chain variable regionCDR1 sequence comprises an amino acid sequence selected from the groupconsisting of amino acid sequences of SEQ ID NOs: 36, 37, 38, 39, 40, 41and 42, and conservative modifications thereof.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families, include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth in (c) through (l) above) usingthe functional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-PD-1 Antibodies of theInvention

In another embodiment, the invention provides antibodies that bind tothe same epitope on human PD-1 as any of the PD-1 monoclonal antibodiesof the invention (i.e., antibodies that have the ability tocross-compete for binding to PD-1 with any of the monoclonal antibodiesof the invention). In preferred embodiments, the reference antibody forcross-competition studies can be the monoclonal antibody 17D8 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs: 1 and 8,respectively), or the monoclonal antibody 2D3 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 2 and 9, respectively), or themonoclonal antibody 4H1 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs: 3 and 10, respectively), or the monoclonal antibody 5C4(having V_(H) and V_(L) sequences as shown in SEQ ID NOs: 4 and 11,respectively), or the monoclonal antibody 4A11 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 5 and 12, or the monoclonal antibody7D3 (having V_(H) and V_(L) sequences as shown in SEQ ID NOs: 6 and 13,or the monoclonal antibody 5F4 (having V_(H) and V_(L) sequences asshown in SEQ ID NOs: 7 and 14, respectively). Such cross-competingantibodies can be identified based on their ability to cross-competewith 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 in standard PD-1 bindingassays. For example, BIAcore analysis, ELISA assays or flow cytometrymay be used to demonstrate cross-competition with the antibodies of thecurrent invention. The ability of a test antibody to inhibit the bindingof, for example, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, to human PD-1demonstrates that the test antibody can compete with 17D8, 2D3, 4H1,5C4, 4A11, 7D3 or 5F4 for binding to human PD-1 and thus binds to thesame epitope on human PD-1 as 17D8, 2D3, 4H1, 5C4, or 4A11. In apreferred embodiment, the antibody that binds to the same epitope onhuman PD-1 as 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 is a humanmonoclonal antibody. Such human monoclonal antibodies can be preparedand isolated as described in the Examples.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See, U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen-binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21, SEQ ID NOs: 22, 23, 24, 25,26, 27 and 28, and SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 sequences comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42, SEQ IDNOs: 43, 44, 45, 46, 47, 48 and 49, and SEQ ID NOs: 50, 51, 52, 53, 54,55 and 56, respectively. Thus, such antibodies contain the V_(H) andV_(L) CDR sequences of monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11,7D3 or 5F4 yet may contain different framework sequences from theseantibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops”J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference. As another example, the germline DNAsequences for human heavy and light chain variable region genes can befound in the GenBank database. For example, the following heavy chaingermline sequences found in the HCo7 HuMAb mouse are available in theaccompanying GenBank accession numbers: 1-69 (NG_(—)0010109,NT_(—)024637 and BC070333), 3-33 (NG_(—)0010109 and NT_(—)024637) and3-7 (NG_(—)0010109 and NT_(—)024637). As another example, the followingheavy chain germline sequences found in the HCo12 HuMAb mouse areavailable in the accompanying GenBank accession numbers: 1-69(NG_(—)0010109, NT_(—)024637 and BC070333), 5-51 (NG_(—)0010109 andNT_(—)024637), 4-34 (NG_(—)0010109 and NT_(—)024637), 3-30.3 (AJ556644)and 3-23 (AJ406678).

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby selected antibodies of the invention, e.g., similar to the V_(H) 3-33framework sequences (SEQ ID NO: 71) and/or the V_(H) 4-39 frameworksequences (SEQ ID NO: 73) and/or the V_(K) L6 framework sequences (SEQID NO: 72) and/or the V_(K) L15 framework sequences (SEQ ID NO: 74) usedby preferred monoclonal antibodies of the invention. The V_(H) CDR1,CDR2, and CDR3 sequences, and the V_(K) CDR1, CDR2, and CDR3 sequences,can be grafted onto framework regions that have the identical sequenceas that found in the germline immunoglobulin gene from which theframework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089, 5,693,762 and6,180,370 to Queen et al).

Another type of variable-region modification is to mutate amino acidresidues within the V_(H) and/or V_(K) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the invention provides isolatedanti-PD-1 monoclonal antibodies, or antigen-binding portions thereof,comprising a heavy chain variable region comprising: (a) a V_(H) CDR1region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 15, 16,17, 18, 19, 20 and 21; (b) a V_(H) CDR2 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 22, 23, 24,25, 26, 27 and 28, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28; (c) a V_(H) CDR3region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 29, 30,31, 32, 33, 34 and 35; (d) a V_(K) CDR1 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 36, 37, 38,39, 40, 41 and 42, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42; (e) a V_(K) CDR2region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 43, 44,45, 46, 47, 48 and 49; and (f) a V_(K) CDR3 region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 50, 51,52, 53, 54, 55 and 56, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

For example, Table 1 below shows a number of amino acid changes in theframework regions of the anti-PD-1 antibodies 17D8, 2D3, 4H1, 5C4, 4A11,7D3 and 5F4 that differ from the heavy chain parent germline sequence.To return one or more of the amino acid residues in the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis.

Amino acid changes may occur in the framework regions of anti-PD-1antibodies that differ from the light chain parent germline sequence.For example, for 17D8, amino acid residue #47 (within FR2) of V_(K) isan isoleucine whereas this residue in the corresponding V_(K) L6germline sequence is a leucine. To return the framework region sequencesto their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis (e.g., residue #47 (residue #13of FR2) of the V_(K) of 17D8 can be “backmutated” from isoleucine toleucine).

As another example, for 4A11, amino acid residue #20 (within FR1) ofV_(K) is a serine whereas this residue in the corresponding V_(K) L15germline sequence is a threonine. To return the framework regionsequences to their germline configuration, for example, residue #20 ofthe V_(K) of 4A11 can be “backmutated” from serine to threonine. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

The alignment of V_(H) regions for 17d8, 2D3, 4H1, 5C4 and 7D3, againstthe parent germline V_(H) 3-33 sequence is shown in FIG. 8. Thealignment of V_(H) regions for 4A11 and 5F4 against the parent germlineV_(H) 4-39 sequence is shown in FIG. 11.

TABLE 1 Modifications to antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and5F4 from the heavy chain germline configuration. Anti-PD-1 Amino acidAmino acid of Original amino acid of Ab position antibody germlineconfiguration 17D8 10 D G 16 G R 27 V F 28 A T 78 M T 93 M V 2D3 10 D G27 L F 30 T S 85 N S 98 T R 4H1 3 Y Q 84 T N 88 V A 98 S R 5C4 21 D S 23K A 27 I F 80 F Y 98 T R 4A11 29 L I 79 Q H 98 V A 7D3 23 T A 24 T A 27I F 70 L I 74 D N 97 V A 98 T R 5F4 23 S T 29 L I 51 A G 77 R K

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasunpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. Nos. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the salinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII:T256A/S298A, S298A/B333A, S298A/K224A and S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6)fucosyltransferase), such that antibodies expressed in the Ms704, Ms705,and Ms709 cell lines lack fucose on their carbohydrates. The Ms704,Ms705, and Ms709 FUT8^(-/-) cell lines were created by the targeteddisruption of the FUT8 gene in CHO/DG44 cells using two replacementvectors (see U.S. Patent Publication No. 20040110704 by Yamane et al.and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 by Hanai et al. describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the alpha 1,6 bond-relatedenzyme. Hanai et al. also describe cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells,with reduced ability to attach fucose to Asn(297)-linked carbohydrates,also resulting in hypofucosylation of antibodies expressed in that hostcell (see also Shields, R. L. et al. (2002) J. Biol. Chem.277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describescell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III(GnTIII) such that antibodies expressed in the engineered cell linesexhibit increased bisecting GlcNac structures which results in increasedADCC activity of the antibodies (see also Umana et al. (1999) Nat.Biotech. 17:176-180). Alternatively, the fucose residues of the antibodymay be cleaved off using a fucosidase enzyme. For example, thefucosidase alpha-L-fucosidase removes fucosyl residues from antibodies(Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivative other proteins, such as mono (C1-C10) alkcoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and BP 0 401 384 by Ishikawa et al.

Methods of Engineering Antibodies

As discussed above, the anti-PD-1 antibodies having V_(H) and V_(K)sequences disclosed herein can be used to create new anti-PD-1antibodies by modifying the V_(H) and/or V_(K) sequences, or theconstant region(s) attached thereto. Thus, in another aspect of theinvention, the structural features of an anti-PD-1 antibody of theinvention, e.g. 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, are used tocreate structurally related anti-PD-1 antibodies that retain at leastone functional property of the antibodies of the invention, such asbinding to human PD-1. For example, one or more CDR regions of 17D8,2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, or mutations thereof, can be combinedrecombinantly with known framework regions and/or other CDRs to createadditional, recombinantly-engineered, anti-PD-1 antibodies of theinvention, as discussed above. Other types of modifications includethose described in the previous section. The starting material for theengineering method is one or more of the V_(H) and/or V_(K) sequencesprovided herein, or one or more CDR regions thereof. To create theengineered antibody, it is not necessary to actually prepare (i.e.,express as a protein) an antibody having one or more of the V_(H) and/orV_(K) sequences provided herein, or one or more CDR regions thereof.Rather, the information contained in the sequence(s) is used as thestarting material to create a “second generation” sequence(s) derivedfrom the original sequence(s) and then the “second generation”sequence(s) is prepared and expressed as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-PD-1 antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 15, 16, 17, 18, 19, 20 and 21, a CDR2 sequence selected from thegroup consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28, and/or aCDR3 sequence selected from the group consisting of SEQ ID NOs: 29, 30,31, 32, 33, 34 and 35; and/or (ii) a light chain variable regionantibody sequence comprising a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42, a CDR2 sequenceselected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48and 49, and/or a CDR3 sequence selected from the group consisting of SEQID NOs: 50, 51, 52, 53, 54, 55 and 56;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the fractional properties of theanti-PD-1 antibodies described herein, which functional propertiesinclude, but are not limited, to:

(a) the antibody binds to human PD-1 with a K_(D) of 1×10⁻⁷ M or less;

(b) the antibody does not substantially bind to human CD28, CTLA-4 orICOS;

(c) the antibody increases T-cell proliferation in an MLR assay;

(d) the antibody increases interferon-gamma production in an MLR assay;

(e) the antibody increases Il-2 secretion in an MLR assay;

(f) the antibody binds to human PD-1 and cynomolgus monkey PD-1;

(g) the antibody inhibits the binding of PD-L1 and/or PD-L2 to PD-1;

(h) the antibody stimulates antigen-specific to memory responses;

(i) the antibody stimulates antibody responses;

(i) the antibody inhibits tumor cell growth in vivo.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., flow cytometry, bindingassays).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-PD-1 antibody coding sequence and the resultingmodified anti-PD-1 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of the invention can be, for example, DNA or RNA and may ormay not contain intronic sequences. In a preferred embodiment, thenucleic acid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules of the invention are those encodingthe VH and VL sequences of the 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4monoclonal antibodies. DNA sequences encoding the VH sequences of 17D8,2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 57, 58, 59,60, 61, 62 and 63, respectively. DNA sequences encoding the VL sequencesof 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 64,65, 66, 67, 68, 69 and 70, respectively.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DMA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be produced bya variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known. In the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et. al.).

In a preferred embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstPD-1 can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHuMAb mice and KM mice™, respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474):856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice™”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-PD-1 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-PD-1 antibodies of the invention. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-PD-1antibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al., U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of the invention,such mice can be immunized with a purified or enriched preparation ofPD-1 antigen and/or recombinant PD-1, or an PD-1 fusion protein, asdescribed by Lonberg, N. et al. (1994) Nature 368(6474): 856-859;Fishwild, D. et al. (1996) Nature Biotechnology 14; 845-851; and PCTPublication WO 98/24884 and WO 01/14424. Preferably, the mice will be6-16 weeks of age upon the first infusion. For example, a purified orrecombinant preparation (5-50 μg) of PD-1 antigen can be used toimmunize the human Ig mice intraperitoneally.

Detailed procedures to generate fully human monoclonal antibodies toPD-1 are described in Example 1 below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-PD-1 human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo12 transgene canbe bred together into a single mouse having two different human heavychain transgenes (HCo7/HCo12). Alternatively or additionally, the KMmouse™ strain can be used, as described in Example 1.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Alternatively, the single cellsuspensions of splenic lymphocytes from immunized mice can be fusedusing an electric field based electrofusion method, using a Cyto Pulselarge chamber cell fusion electroporator (Cyto Pulse Sciences, Inc.,Glen Burnie, Md.). Cells are plated at approximately 2×10⁵ in flatbottom microliter plate, followed by a two week incubation in selectivemedium containing 20% fetal Clone Serum, 18% “653” conditioned media, 5%origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After approximately two weeks, cells can be cultured in mediumin which the HAT is replaced with HT. Individual, wells can then bescreened by ELISA for human monoclonal IgM and IgG antibodies. Onceextensive hybridoma growth occurs, medium can be observed usually after10-14 days. The antibody secreting hybridomas can be replated, screenedagain, and if still positive for human IgG, the monoclonal antibodiescan be subcloned at least twice by limiting dilution. The stablesubclones can then be cultured in vitro to generate small amounts ofantibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N. J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealliquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of theInvention

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(K) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic of eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1995) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Characterization of Antibody binding to Antigen

Antibodies of the invention can be tested for binding to PD-1 by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified PD-1 at 0.25 μg/ml in PBS, and then blocked with 5% bovineserum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasmafrom PD-1-immunized mice) are added to each well and incubated for 1-2hours at 37° C. The plates are washed with PBS/Tween and then incubatedwith secondary reagent (e.g., for human antibodies, a goat-anti-humanIgG Fc-specific polyclonal reagent) conjugated to alkaline phosphatasefor 1 hour at 37° C. After washing, the plates are developed with pNPPsubstrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, micewhich develop the highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with PD-1 immunogen. Hybridomasthat bind with high avidity to PD-1 are subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-PD-1 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N. J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-PD-1 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using PD-1 coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISA can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-PD-1 human IgGs can be further tested for reactivity with PD-1antigen by Western blotting. Briefly, PD-1 can be prepared and subjectedto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% total calf serum, and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St Louis, Mo.).

Immunoconjugates

In another aspect, the present invention features an anti-PD-1 antibody,or a fragment thereof, conjugated to a therapeutic moiety, such as acytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Suchconjugates are referred to herein as “immunoconjugates”.Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly antinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cyctotoxins that can beconjugated to an antibody of the invention include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg™; Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins (B, C,D).

For further discussion of types of endotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparing radioimmunoconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (IDEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies forImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan, R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84;Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-PD-1 antibody, or a fragment thereof, of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may in fact be derivatized or linkd to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthe invention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for PD-1 and a secondbinding specificity for a second target epitope. In a particularembodiment of the invention, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, the invention includes bispecific molecules capable ofbinding both to FcγR or FcαR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)), and to target cellsexpressing PD-1. These bispecific molecules target PD-1 expressing cellsto effector cell and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of an PD-1 expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion.

In an embodiment of the invention in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-PD-1 binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the F_(C) receptor or target cellantigen. The “anti-enhancement factor portion” can bind an F_(C)receptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell thatresults in an increased immune response against the target cell).

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes; FcγRI(CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72kDa molecule, which shows high affinity for monomeric IgG (10⁸-10⁹M⁻¹).

The production and characterization of certain preferred anti-Fcγmonoclonal antibodies are described by Fanger et al. in PCT PublicationWO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from theFc γ binding site of the receptor and, thus, their binding is notblocked substantially by physiological levels of IgG. Specificanti-FcγRI antibodies useful in this invention are mAb 22, mAb 32, mAb44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is available fromthe American Type Culture Collection, ATCC Accession No. HB9469. Inother embodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol.155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibodyproducing cell line was deposited at the American Type CultureCollection under the designation HA022CL1 and has the accession no. CRL11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constituitively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-GSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992), J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of the invention because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); (4) mediate enhanced antigen presentation of antigens,including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the invention are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-PD-1 binding specificities, using methods known in the art. Forexample, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139:2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent, (e.g., an antibody) specificfor the complex of interest. For example, the FcR-antibody complexes canbe detected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-PD-1 antibody of the presentinvention combined with at least one other anti-inflammatory orimmunosuppressant agent. Examples of therapeutic agents that can be usedin combination therapy are described in greater detail below in thesection on uses of the antibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e, antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred per cent, this amount will range from about 0.01 per centto about ninety-nine percent of active ingredient, preferably from about0.1 per cent to about 70 per cent, most preferably from about 1 percent, to about 30 per cent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-PD-1antibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-PD-1 antibody of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumors, a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth can be evaluated in an animal model system,predictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected.

In another aspect, the instant disclosure provides a pharmaceutical kitof parts comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody,as described herein. The kit may also further comprise instructions foruse in the treatment of a hyperproliferative disease (such as cancer asdescribed herein). In another embodiment, the anti-PD-1 and anti-CTLA-1antibodies may be co-packaged in unit dosage form.

In certain embodiments, two or more monoclonal antibodies with differentbinding specificities (e.g., anti-PD-1 and anti-CTLA-4) are administeredsimultaneously, in which case the dosage of each antibody administeredfalls within the ranges indicated. Antibody can be administered as asingle dose or more commonly can be administered on multiple occasions.Intervals between single dosages can be, for example, weekly, monthly,every three months or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody to the target antigen inthe patient. In some methods, dosage is adjusted to achieve a plasmaantibody concentration of about 1-1000 μg/ml and in some methods about25-300 μg/ml.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811, 5,374,548, and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat, No. 5,416,016 to Low et al.);mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanan; M. L. Laukkanan (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies, antibody compositions and methods of the presentinvention have numerous in vitro and in vivo utilities involving, forexample, detection of PD-1 or enhancement of immune response by blockadeof PD-1. In a preferred embodiment the antibodies of the presentinvention are human antibodies. For example, these molecules can beadministered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to enhance immunity in a variety of situations.Accordingly, in one aspect, the invention provides a method of modifyingan immune response in a subject comprising administering to the subjectthe antibody, or antigen-binding portion thereof, of the invention suchthat the immune response in the subject is modified. Preferably, theresponse is enhanced, stimulated or up-regulated.

As used herein, the term “subject” is intended to include human andnon-human animals. Non-human animals includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dogs, cats,cows, horses, chickens, amphibians, and reptiles, although mammals arepreferred, such as non-human primates, sheep, dogs, cats, cows andhorses. Preferred subjects include human patients in need of enhancementof an immune response. The methods are particularly suitable fortreating human patients having a disorder that can be treated byaugmenting the T-cell mediated immune response. In a particularembodiment, the methods are particularly suitable for treatment ofcancer cells in vivo. To achieve antigen-specific enhancement ofimmunity, the anti-PD-1 antibodies can be administered together with anantigen of interest. When antibodies to PD-1 are administered togetherwith another agent, the two can be administered in either order orsimultaneously.

The invention further provides methods for detecting the presence ofhuman PD-1 antigen in a sample, or measuring the amount of human PD-1antigen, comprising contacting the sample, and a control sample, with ahuman monoclonal antibody, or an antigen-binding portion thereof, whichspecifically binds to human PD-1, under conditions that allow forformation of a complex between the antibody or portion thereof and humanPD-1. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of human PD-1 antigen in the sample.

Given the specific binding of the antibodies of the invention for PD-1,compared to CD28, ICOS and CTLA-4, the antibodies of the invention canbe used to specifically detect PD-1 expression on the surface of cellsand, moreover, can be used to purify PD-1 via immunoaffinitypurification.

Cancer

Blockade of PD-1 by antibodies can enhance the immune response tocancerous cells in the patient. The ligand for PD-1, PD-L1, is notexpressed in normal human cells, but is abundant in a variety of humancancers (Dong et al. (2002) Nat Med 8:787-9). The interaction betweenPD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes,a decrease in T-cell receptor mediated proliferation, and immune evasionby the cancerous cells (Dong et al. (2003) J Mol Med 81:281-7; Blank etal. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004)Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed byinhibiting the local interaction of PD-1 to PD-L1 and the effect isadditive when the interaction of PD-1 to PD-L2 is blocked as well (Iwaiet al. (2002) PNAS 99:12293-7; Brown et al. (2003) J. Immunol.170:1257-66). While previous studies have shown that T-cellproliferation can be restored by inhibiting the interaction of PD-1 toPD-L1, there have been no reports of a direct effect on cancer tumorgrowth in vivo by blocking the PD-1/PD-L1 interaction. In one aspect,the present invention relates to treatment of a subject in vivo using ananti-PD-1 antibody such that growth of cancerous tumors is inhibited. Ananti-PD-1 antibody may be used alone to inhibit the growth of canceroustumors. Alternatively, an anti-PD-1 antibody may be used in conjunctionwith other immunogenic agents, standard cancer treatments, or otherantibodies, as described below.

Accordingly, in one embodiment, the invention provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-PD-1antibody, or antigen-binding portion thereof. Preferably, the antibodyis a human anti-PD-1 antibody (such as any of the human anti-human PD-1antibodies described herein). Additionally or alternatively, theantibody may be a chimeric or humanized anti-PD-1 antibody.

Preferred cancers whose growth may be inhibited using the antibodies ofthe invention include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment includemelanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clearcell carcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), breast cancer, colon cancer and lung cancer (e.g.non-small cell lung cancer). Additionally, the invention includesrefractory or recurrent malignancies whose growth may be inhibited usingthe antibodies of the invention.

Examples of other cancers that may be treated using the methods of theinvention include bone cancer, pancreatic cancer, skin cancer, cancer ofthe head or neck, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, chronic oracute leukemias including acute myeloid leukemia, chronic myeloidleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder,cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasmof the central nervous system (CNS), primary CNS lymphoma, tumorangiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-celllymphoma, environmentally induced cancers including those induced byasbestos, and combinations of said cancers. The present invention isalso useful for treatment of metastatic cancers, especially metastaticcancers that express PD-L1 (Iwai et al. (2005) Int. Immunol.17:133-144).

Optionally, antibodies to PD-1 can be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transferred to express the cytokine GM-CSF (discussedfurther below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. It is anticipated that by raising the threshold of T cellactuation by PD-1 blockade, we may expect to activate tumor responses inthe host.

PD-1 blockade is likely to be most effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., 2000, Developmentof Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C.,2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCOEducational Book Spring: 414-428; Foon, K. 2000, ASCO Educational BookSpring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines,Ch. 61, pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, Cancer:Principles and Practice of Oncology, Fifth Edition). In one of thesestrategies, a vaccine is prepared using autologous or allogeneic tumorcells. These cellular vaccines have been shown to be most effective whenthe tumor cells are transduced to express GM-CSF. GM-CSF has been shownto be a potent activator of antigen presentation for tumor vaccination(Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90:3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. PD-1 blockade may be used in conjunction witha collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim, N et al. (1994)Science 266: 2011-2013). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (ie. bcr-abl in the Philadelphia chromosome), oridiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PD-1blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot, R &Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997)Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al. (1998) Nature Medicine 4:328-332). DCsmay also be transduced, by genetic means to express these tumor antigensas well. DCs have also been fused directly to tumor cells for thepurposes of immunization (Kugler, A. et al. (2000) Nature Medicine6:332-336). As a method of vaccination, DC immunization may beeffectively combined with PD-1 blockade to activate more potentanti-tumor responses.

PD-1 blockade may also be combined with standard cancer treatments. PD-1blockade may be effectively combined with chemotherapeutic regimes. Inthese instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr, M. et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is ananti-PD-1 antibody in combination with decarbazine for the treatment ofmelanoma. Another example of such a combination is as anti-PD-1 antibodyin combination with interleukin-2 (IL-2) for the treatment of melanoma.The scientific rationale behind the combined use of PD-1 blockade andchemotherapy is that cell death, that is a consequence of the cytotoxicaction of most chemotherapeutic compounds, should result in increasedlevels of tumor antigen in the antigen presentation pathway. Othercombination therapies that may result in synergy with PD-1 blockadethrough cell death are radiation, surgery, and hormone deprivation. Eachof these protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors may also be combined with PD-1 blockade.Inhibition of angiogenesis leads to tumor cell death which may feedtumor antigen into host antigen presentation pathways.

PD-1 blocking antibodies can also be used in combination with bispecificantibodies that target Fc alpha or Fc gamma receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would by augmentedby the use of PD-1 blockade. Alternatively, antigen may be delivereddirectly to DCs by the use of bispecific antibodies which bind to tumorantigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al. (1996) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies to each of these entities may be used incombination with anti-PD-1 to counteract the effects of theimmunosuppressive agent and favor tumor immune responses by the host.

Other antibodies which may be used to activate host immuneresponsiveness can be used in combination with anti-PD-1. These includemolecules on the surface of dendritic cells which activate DC functionand antigen presentation. Anti-CD40 antibodies are able to substituteeffectively for T cell helper activity (Ridge, J. et al. (1998) Nature393: 474-478) and can be used in conjunction with PD-1 antibodies (Ito,N. et al. (2000) Immunobiology 201 (5) 527-40). Activating antibodies toT cell costimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No.5,811,097), OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169),4-1BB (Melero, I. et al. (1997) Nature Medicine 3: 682-685 (1997), andICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) may also providefor increased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. PD-1 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) Science 285:546-51). These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofanti-PD-1 antibodies may be expected to increase the frequency andactivity of the adoptively transferred T cells.

Infectious diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. Accordingly, another aspectof the invention provides a method of treating an infectious disease ina subject comprising administering to the subject an anti-PD-1 antibody,or antigen-binding portion thereof, such that the subject is treated forthe infectious disease. Preferably, the antibody is a human anti-humanPD-1 antibody (such as any of the human anti-PD-1 antibodies describedherein). Additionally or alternatively, the antibody can be a chimericor humanized antibody.

Similar to its application to tumors as discussed above, antibodymediated PD-1 blockade can be used alone, or as an adjuvant, incombination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. Examples of pathogens for whichthis therapeutic approach may be particularly useful, include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, & C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, PseodomonasAeruginosa. PD-1 blockade is particularly useful against establishedinfections by agents such as HIV that present altered antigens over thecourse of the infections. These novel epitopes are recognized as foreignat the time of anti-human PD-1 administration, thus provoking a strong Tcell response that is not dampened by negative signals through PD-1.

Some examples of pathogenic viruses causing infections treatable bymethods of the invention include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusel, glabrata,topicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.). Genus Mucarales (mucor, absidia, rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, and Nippostrongylus brasilieasis.

In all of the above methods, PD-1 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

Autoimmune Reactions

Anti-PD-1 antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (depigmentation observed in anti-CTLA-4+GM-CSF-modified B16melanoma in van Elsas et al. supra; depigmentation in Trp-2 vaccinatedmice (Overwijk, W. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2982-2987); antoimmune prostatitis evoked by TRAMP tumor cell vaccines(Hurwitz, A. (2000) supra), melanoma peptide antigen vaccination andvitilago observed in human clinical trials (Rosenberg, S A and White, DE (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4).

Therefore, it is possible to consider using anti-PD-1 blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimers disease involvesinappropriate accumulation of Aβ peptide in amyloid deposits in thebrain; antibody responses against amyloid are able to clear theseamyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins may also be used as targets such as IgE for thetreatment of allergy and asthma, and TNFα for rhematoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-PD-1 antibody. Neutralizing antibody responses toreproductive hormones may be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors may also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-PD-1 antibodycan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including Aβ in Alzheimer's disease, cytokinessuch as TNFα, and IgE.

Vaccines

Anti-PD-1 antibodies may be used to stimulate antigen-specific immuneresponses by coadministration of an anti-PD-1 antibody with an antigenof interest (e.g., a vaccine). Accordingly, in another aspect theinvention provides a method of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-PD-1 antibody, or antigen-binding portionthereof, such that an immune response to the antigen in the subject isenhanced. Preferably, the antibody is a human anti-human PD-1 antibody(such as any of the human anti-PD-1 antibodies described herein).Additionally or alternatively, the antibody can be a chimeric orhumanized antibody. The antigen can be, for example, a tumor antigen, aviral antigen, a bacterial antigen or an antigen from a pathogen.Non-limiting examples of such antigens include those discussed in thesections above, such as the tumor antigens (or tumor vaccines) discussedabove, or antigens from the viruses, bacteria or other pathogensdescribed above.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of the invention in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-PD-1 antibodies of the invention canbe co-administered with one or other more therapeutic agents, e.g., acytotoxic agent a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent, (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin, sulfate, carmustins,chlorambucil, decarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/dose onceevery four weeks and adriamycin is intravenously administered as a 60-75mg/ml dose once every 21 days. Co-administration of the human anti-PD-1antibodies, or antigen binding fragments thereof, of the presentinvention with chemotherapeutic agents provides two anti-cancer agentswhich operate via different mechanisms which yield a cytotoxic effect tohuman tumor cells. Such co-administration can solve problems due todevelopment of resistance to drugs or a change in the antigenicity ofthe tumor cells which would render them unreactive with the antibody.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention (e.g., human antibodies,bispecific or multispecific molecules, or immunoconjugates) andinstructions for use. The kit can further contain a least one additionalreagent, or one or more additional human antibodies of the invention(e.g., a human antibody having a complementary activity which binds toan epitope in PD-1 antigen distinct from the first human antibody). Kitstypically include a label indicating the intended use of the contents ofthe kit. The term label includes any writing, or recorded materialsupplied on or with the kit, or which otherwise accompanies the kit.

Combination Therapy

The present invention is based, in part, on the following experimentaldata. Mouse tumor models (MC38 colon cancer and SA1/N fibrosarcoma) wereused to examine the in vivo effect of treating a tumor by combiningimmunostimulatory therapeutic antibodies—anti-CTLA-1 and anti-PD-1. Theimmunotherapeutic combination was provided either simultaneous with theimplant of tumor cells (Examples 14 and 17) or after the tumor cellswere implanted for a time sufficient to become an established tumor(Examples 15, 16 and 18). Regardless of the timing of antibodytreatment, it was found that anti-CTLA-4 antibody treatment alone andanti-PD-1 antibody (chimeric antibody in which a rat anti-mouse PD-1 wasmodified with a mouse immunoglobulin Fc region, see Example 1) treatmentalone had a modest effect on reducing tumor growth in the MC38 tumormodel (see, e.g., FIGS. 21, 24 and 27). The anti-CTLA-4 antibody alonewas quite effective in the SA1/N tumor model (see FIG. 30D), whichrequired a lower anti-CTLA-4 antibody dose for the combination studiesin this model. Nonetheless, the combination, treatment of anti-CTLA-4antibody and anti-PD-1 antibody showed an unexpected, significantlygreater effect on reducing tumor growth as compared to treatment witheither antibody alone (see, e.g., FIGS. 21D, 24D, 30F and 33H-J). Inaddition, the results of Examples 14, 16 and 18 show that thecombination treatment of anti-CTLA-4 antibody and anti-PD-1 antibody hada significant (synergistic) effect on tumor growth even at sub-optimaltherapeutic doses as compared to treatment with either antibody alone(i.e., the combination therapy was surprisingly more effective atsubtherapeutic doses than either monotherapy). Without wishing to bebound by theory, it is possible that by raising the threshold of T cellactivation by PD-1 and CTLA-4 blockade, anti-tumor responses may beactivated in a host.

In one embodiment, the present invention provides a method for treatinga hyperproliferative disease, comprising administering a PD-1 antibodyand a CTLA-4 antibody to a subject. In further embodiments, theanti-PD-1 antibody is administered at a subtherapeutic dose, theanti-CTLA-4 antibody is administered at a subtherapeutic dose, or bothare administered at a subtherapeutic dose. In another embodiment, thepresent invention provides a method for altering an adverse eventassociated with treatment of a hyperproliferative disease with animmunostimulatory agent, comprising administering an anti-PD-1 antibodyand a subtherapeutic dose of anti-CTLA-4 antibody to a subject. Incertain embodiments, the subject is human. In certain embodiments, theanti-CTLA-4 antibody is human sequence monoclonal antibody I0D1 and theanti-PD-1 antibody is human sequence monoclonal antibody, such as 17D8,2D3, 4H1, 5C4 and 4A11. Human sequence monoclonal antibodies 17D8, 2D3,4H1, 5C4 and 4A11 have been isolated and structurally characterized, asdescribed in U.S. Provisional Patent No. 60/679,466.

The anti-CTLA-4 antibody and anti-PD-1 monoclonal antibodies (mAbs) andthe human sequence antibodies of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256:495. Any technique for producingmonoclonal antibody can be employed, e.g., viral or oncogenictransformation of B lymphocytes. One animal system for preparinghybridomas is the murine system. Hybridoma production in the mouse is avery well-established procedure. Immunization protocols and techniquestor isolation of immunized splenocytes for fusion are known in the art.Fusion partners (e.g., murine myeloma cells) and fusion procedures arealso known (see, e.g., Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.).

Anti-CTLA-4 antibodies of the instant invention can bind to an epitopeon human CTLA-4 so as to inhibit CTLA-4 from interacting with a human B7counterreceptor. Because interaction of human CTLA-4 with human B7transduces a signal leading to inactivation of T-cells bearing the humanCTLA-4 receptor, antagonism of the interaction effectively induces,augments or prolongs the activation of T cells bearing the human CTLA-4receptor, thereby prolonging or augmenting an immune response.Anti-CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097;5,855,887; 6,051,227; in PCT Application Publication Nos. WO 01/14424and WO 00/37504; and in U.S. Patent Publication No. 2002/0039581. Eachof these references is specifically incorporated herein by reference forpurposes of description of anti-CTLA-4 antibodies An exemplary clinicalanti-CTLA-4 antibody is human monoclonal antibody 10D1 as disclosed inWO 01/14424 and U.S. patent application Ser. No. 09/644,668. Antibody10D1 has been administered in single and multiple doses, alone or incombination with a vaccine, chemotherapy, or interleukin-2 to more than500 patients diagnosed with metastatic melanoma, prostate cancer,lymphoma, renal cell cancer, breast cancer, ovarian cancer, and HIV.Other anti-CTLA-4 antibodies encompassed by the methods of the presentinvention include, for example, those disclosed in WO 98/42752; WO00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl.Acad. Sci. USA 95( 17)10067-10071; Camacho et al. (2004) J. Clin.Oncology 22(145). Abstract No. 2505 (antibody CP-675206): and Mokyr etal. (1998) Cancer Res. 58:5301-5304. In certain embodiments, the methodsof the instant invention comprise use of an anti-CTLA-4 antibody that isa human sequence antibody, preferably a monoclonal antibody and inanother embodiment is monoclonal antibody 10D1.

In certain embodiments, the anti-CTLA-4 antibody binds to human CTLA-4with a K_(D) of 5×10⁻⁸ M or less, binds to human CTLA-4 with a K_(D) of1×10⁻⁸ M or less, binds to human CTLA-4 with a K_(D) of 5×10⁻⁹ M orless, or binds to human CTLA-4 with a K_(D) of between 1×10⁻⁸ M and1×10⁻¹⁰ M or less.

The combination of antibodies is useful for enhancement of an immuneresponse against a hyperproliferative disease by blockade of PD-1 andCTLA-4. In a preferred embodiment, the antibodies of the presentinvention are human antibodies. For example, these molecules can beadministered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to enhance immunity in a variety of situations.Accordingly, in one aspect, the invention provides a method of modifyingan immune response in a subject comprising administering to the subjectan antibody combination, or a combination of antigen-binding portionsthereof, of the invention such that the immune response in the subjectis modified. Preferably, the response is enhanced, stimulated orup-regulated. In another embodiment, the instant disclosure provides amethod of altering adverse events associated with treatment of ahyperproliferative disease with an immunostimulatory therapeutic agent,comprising administering an anti-PD1 antibody and a subtherapeutic doseof anti-CTLA-4 antibody to a subject.

Blockade of PD-1 and CTLA-4 by antibodies can enhance the immuneresponse to cancerous cells in the patient. Cancers whose growth may beinhibited using the antibodies of the instant disclosure include cancerstypically responsive to immunotherapy. Representative examples ofcancers for treatment with the combination therapy of the instantdisclosure include melanoma (e.g., metastatic malignant melanoma), renalcancer, prostate cancer, breast cancer, colon cancer and lung cancer.Examples of other cancers that may be treated using the methods of theinstant disclosure include bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, testicular cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,chronic or acute leukemias including acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of thebladder, cancer of the kidney or ureter, carcinoma of the renal pelvis,neoplasm of the central nervous system (CNS), primary CNS lymphoma,tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,T-cell lymphoma, environmentally induced cancers including those inducedby asbestos, and combinations of said cancers. The present invention isalso useful for treatment of metastatic cancers.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein may be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier, or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. For example, ananti-CTLA-4 antibody and an anti-PD-1 antibody can be administeredsequentially, such as anti-CTLA-4 being administered first and anti-PD-1second, or anti-PD-1 being administered first and anti-CTLA-4 second.Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations may be combined withconcurrent administrations, or any combination thereof. For example, thefirst administration of a combination anti-CTLA-4 antibody and anti-PD-1antibody may be concurrent, the second administration may be sequentialwith anti-CTLA-4 first and anti-PD-1 second, and the thirdadministration may be sequential with anti-PD-1 first and anti-CTLA-4second, etc. Another representative dosing scheme may involve a firstadministration that is sequential with anti-PD-1 first and anti-CTLA-4second, and subsequent administrations may be concurrent.

Optionally, the combination of anti-PD-1 and anti-CTLA-4 antibodies canbe further combined with an immunogenic agent, such as cancerous cells,purified tumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines (He et al. (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gp100, MAGEantigens, Trp-1 MART1 and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF (discussed further below).

A combined PD-1 and CTLA-4 blockade can be further combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S. (2000) Developmentof Cancer Vaccines, ASCO Educational Book Spring: 66-62; Logothetis, C.,2000, ASCO Educational Book Spring: 300-302; Khayat, D. (2000) ASCOEducational Book Spring: 414-428; Foon, K. (2000) ASCO Educational BookSpring: 730-738; see also Restifo and Sznol, Cancer Vaccines, Ch. 61,pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles andPractice of Oncology, Fifth Edition). In one of these strategies, avaccine is prepared using autologous or allogeneic tumor cells. Thesecellular vaccines have been shown to be most effective when the tumorcells are transduced to express GM-CSF. GM-CSF has been shown to be apotent activator of antigen presentation for tumor vaccination (Dranoffet al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg (1999) Immunity 10:281-7). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. In certain embodiments, a combined PD-1 andCTLA-4 blockade using the antibody compositions described herein may beused in conjunction with a collection of recombinant proteins and/orpeptides expressed in a tumor in order to generate an immune response tothese proteins. These proteins are normally viewed by the immune systemas self-antigens and are, therefore, tolerant to them. The tumor antigenmay also include the protein telomerase, which is required for thesynthesis of telomeres of chromosomes and which is expressed in morethan 85% of human cancers and in only a limited number of somatictissues (Kim et al. (1994) Science 266: 2011-2013). (These somatictissues may be protected from immune attack by various means). Tumorantigen may also be “neo-antigens” expressed in cancer cells because ofsomatic mutations that alter protein sequence or create fusion proteinsbetween two unrelated sequences (i.e., bcr-abl in the Philadelphiachromosome), or idiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PD-1blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4:328-332). DCs mayalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization may be effectively furthercombined with a combined PD-1 and CTLA-4 blockade to activate morepotent anti-tumor responses.

A combined PD-1 and CTLA-4 blockade may also be further combined withstandard cancer treatments. For example, a combined PD-1 and CTLA-4blockade may be effectively combined with chemotherapeutic regimes. Inthese instances, as is observed with the combination of anti-PD-1 andanti-CTLA-4 antibodies, it may be possible to reduce the dose of otherchemotherapeutic reagent administered with the combination of theinstant disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304).An example of such a combination is a combination of anti-PD-1 andanti-CTLA-4 antibodies further in combination with decarbazine for thetreatment of melanoma. Another example is a combination of anti-PD-1 andanti-CTLA-4 antibodies further in combination with interleukin-2 (IL-2)for the treatment of melanoma. The scientific rationale behind thecombined use of PD-1 and CTLA-4 blockade with chemotherapy is that celldeath, which is a consequence of the cytotoxic action of mostchemotherapeutic compounds, should result in increased levels of tumorantigen in the antigen presentation pathway. Other combination therapiesthat may result in synergy with a combined PD-1 and CTLA-4 blockadethrough cell death include radiation, surgery, or hormone deprivation.Each of these protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors may also be combined with a combined PD-1 andCTLA-4 blockade. Inhibition of angiogenesis leads to tumor cell death,which may also be a source of tumor antigen to be fed into host antigenpresentation pathways.

A combination of PD-1 and CTLA-4 blocking antibodies can also be used incombination with bispecific antibodies that target Fcα or Fcγreceptor-expressing effector cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. Far example anti-Fc receptor/anti tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. The T cell arm of these responseswould by augmented by the use of a combined PD-1 and CTLA-4 blockade.Alternatively, antigen may be delivered directly to DCs by the use ofbispecific antibodies which bind to tumor antigen and a dendritic cellspecific cell surface marker.

In another example, a combination of anti-PD-1 and anti-CTLA-4antibodies can be used in conjunction with anti-neoplastic antibodies,such as Rituxan®0 (rituximab), Herceptin® (trastuzumab), Bexxar®(tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab),Lymphocide® (eprtuzumab), Avastin® (bevacizumab), and Tarceva®(erlotinib), and the like. By way of example and not wishing to be boundby theory, treatment with an anti-cancer antibody or an anti-cancerantibody conjugated to a toxin can lead to cancer cell death (e.g.,tumor cells) which would potentiate an immune response mediated byCTLA-4 or PD-1. In an exemplary embodiment, a treatment of ahyperproliferative disease (e.g., a cancer tumor) may include ananticancer antibody in combination with anti-PD-1 and anti-CTLA-4antibodies, concurrently or sequentially or any combination thereof,which may potentiate an anti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-β (Kehrl, J. et al.(1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. & O'Garra, A.(1992) Immunology Today 13: 198-200), and Fas ligand (Hahne, M. et al.(1996) Science 274: 1363-1365). In another example, antibodies to eachof these entities may be further combined with an anti-PD-1 andanti-CTLA-4 combination to counteract the effects of immunosuppressiveagents and favor anti-tumor immune responses by the host.

Other antibodies that may be used to activate host immune responsivenesscan be further used in combination with an anti-PD-1 and anti-CTLA-4combination. These include molecules on the surface of dendritic cellsthat activate DC function and antigen presentation. Anti-CD40 antibodiesare able to substitute effectively for T cell helper activity (Ridge, J.et al. (1998) Nature 393: 474-478) and can be used in conjunction withan anti-PD-1 and anti-CTLA-4 combination (Ito, N. et al. (2000)Immunobiology 201 (5) 527-40). Activating antibodies to T cellcostimulatory molecules, such as OX-40 (Weinberg, A. et al. (2000)Immunol 164: 2160-2169), 4-IBB (Melero, I. et al. (1997) Nature Medicine3: 682-685 (1997), and ICOS (Hutloff, A. et al. (1999) Nature 397:262-266) may also provide for increased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. A combined PD-1 and CTLA-4 blockade can beused to increase the effectiveness of the donor engrafted tumor specificT cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) Science 285:546-51). These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofanti-PD-4 and anti-CTLA-4 antibodies may be expected to increase thefrequency and activity of the adoptively transferred T cells.

As set forth herein, organs can exhibit immune-related adverse eventsfollowing immunostimulatory therapeutic antibody therapy, such as the GItract (diarrhea and colitis) and the skin (rash and pruritis) aftertreatment with anti-CTLA-4 antibody. For example, non-colonicgastrointestinal immune-related adverse events have also been observedin the esophagus (esophagitis), duodenum (duodenitis), and Ileum(ileitis) after anti-CTLA-4 antibody treatment.

In certain embodiments, the present invention provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering a anti-PD-1 antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject. For example, the methods of thepresent invention provide for a method of reducing the incidence ofimmunostimulatory therapeutic antibody-induced colitis or diarrhea byadministering a non-absorbable steroid to the patient. Because anypatient who will receive an immunostimulatory therapeutic antibody is atrisk for developing colitis or diarrhea induced by such an antibody,this entire patient population is suitable for therapy according to themethods of the present invention. Although steroids have beenadministered to treat inflammatory bowel disease (IBD) and preventexacerbations of IBD, they have not been used to prevent (decrease theincidence of) IBD in patients who have not been diagnosed with IBD. Thesignificant side effects associated with steroids, even non-absorbablesteroids, have discouraged prophylactic use.

In further embodiments, a combination PD-1 and CTLA-4 blockade (i.e.,immunostimulatory therapeutic antibodies anti-PD-1 and anti-CTLA-4) canbe further combined with the use of any non-absorbable steroid. As usedherein, a “non-absorbable steroid” is a glucocorticoid that exhibitsextensive first pass metabolism such that, following metabolism in theliver, the bioavailability of the steroid is low, i.e., less than about20%. In one embodiment of the invention, the non-absorbable steroid isbudesonide. Budesonide is a locally-acting glucocorticosteroid, which isextensively metabolized, primarily by the liver, following oraladministration. ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependentoral formulation of budesonide developed to optimize drag delivery tothe ileum and throughout the colon. ENTOCORT EC® is approved in the U.S.for the treatment of mild to moderate Crohn's disease involving theileum and/or ascending colon. The usual oral dosage of ENTOCORT EC® forthe treatment of Crohn's disease is 6 to 9 mg/day. ENTOCORT EC® isreleased in the intestines before being absorbed and retained in the gutmucosa. Once it passes through the gut mucosa target tissue, ENTOCORTEC® is extensively metabolized by the cytochrome P450 system in theliver to metabolites with negligible glucocorticoid activity. Therefore,the bioavailability is low (about 10%). The low bioavailability ofbudesonide results in an improved therapeutic ratio compared to otherglucocorticoids with less extensive first-pass metabolism. Budesonideresults in fewer adverse effects, including less hypothalmic-pituitarysuppression, than systemically-acting corticosteroids. However, chronicadministration of ENTOCORT EC® can result in systemic glucocorticoideffects such as hypercorticism and adrenal suppression. See PDR 58^(th)ed. 2004; 608-610.

In still further embodiments, a combination PD-1 and CTLA-4 blockade(i.e., immunostimulatory therapeutic antibodies anti-PD-1 andanti-CTLA-4) in conjunction with a non-absorbable steroid can be furthercombined with a salicylate. Salicylates include 5-ASA agents such as,for example: sulfasalazine (AZULFIDINE®, Pharmacia & UpJohn); olsalazine(DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, SalixPharmaceuticals, Inc.); and mesalamime (ASACOL®, Procter & GamblePharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.;ROWASA®, Solvay).

In accordance with the methods of the present invention, a salicylateadministered in combination with anti-PD-1 and anti-CTLA-4 antibodiesand a non-absorbable steroid can includes any overlapping or sequentialadministration of the salicylate and the non-absorbable steroid for thepurpose of decreasing the incidence of colitis induced by theimmunostimulatory antibodies. Thus, for example, methods for reducingthe incidence of colitis induced by the immunostimulatory antibodiesaccording to the present invention encompass administering a salicylateand a non-absorbable concurrently or sequentially (e.g., a salicylate isadministered 6 hours after a non-absorbable steroid), or any combinationthereof. Further, according to the present invention, a salicylate and anon-absorbable steroid can be administered by the same route (e.g., bothare administered orally) or by different routes (e.g., a salicylate isadministered orally and a non-absorbable steroid is administeredrectally), which may differ from the route(s) used to administer theanti-PD-1 and anti-CTLA-4 antibodies.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Monoclonal Antibodies AgainstPD-1 Antigen

Immunization protocol utilized as antigen both (i) a recombinant fusionprotein comprising the extracellular portion of PD-1 and (ii) membranebound full-length PD-1. Both antigens were generated by recombinanttransfection methods in a CHO cell line.

Transgenic HuMab and KM Mice™

Fully human monoclonal antibodies to PD-1 were prepared using the HCo7strain of HuMab transgenic mice and the KM strain of transgenictranschromosomic mice, each of which express human antibody genes. Ineach of these mouse strains, the endogenous mouse kappa light chain genehas been homozygously disrupted as described in Chen et al. (1993) EMBOJ. 12:811-820 and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of PCT Publication WO01/09187. Each of these mouse strains carries a human kappa light chaintransgenic, KCo5, as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851. The HCo7 strain carries the HCo7 human heavychain transgene as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and5,545,807. The KM strain contains the SC20 transchromosome as describedin PCT Publication WO 02/43478.

HuMab and KM Immunizations:

To generate fully human monoclonal antibodies to PD-1, HuMab mice and KMmice™ were immunized with purified recombinant PD-1 fusion protein andPD-1-transfected CHO cells as antigen. General immunization schemes forHuMab mice are described in Lonberg, N. et al. (1994) Nature 368(6474):856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 andPCT Publication WO 98/24884. The mice were 6-16 weeks of age upon thefirst infusion of antigen. A purified recombinant preparation (5-50 μg)of PD-1 fusion protein antigen and 5-10×10⁶ cells were used to immunizethe HuMab mice and KM mice™ intraperitonealy, subcutaneously (Sc) or viafootpad injection.

Transgenic mice were immunized twice with antigen in complete Freund'sadjuvant or Ribi adjuvant IP, followed by 3-21 days IP (up to a total of11 immunizations) with the antigen in incomplete Freund's or Ribiadjuvant. The immune response was monitored by retroorbital bleeds. Theplasma was screened by ELISA (as described below), and mice withsufficient titers of anti-PD-1 human immunogolobulin were used forfusions. Mice were boosted intravenously with antigen 3 days beforesacrifice and removal of the spleen. Typically, 10-35 fusions for eachantigen were performed. Several dozen mice were immunized for eachantigen.

Selection of HuMab or KM Mice™ Producing Anti-PD-1 Antibodies:

To select HuMab or KM mice™ producing antibodies that bound PD-1, serafrom immunized mice were tested by ELISA as described by Fishwild, D. etal. (1996). Briefly, microtiter plates were coated with purifiedrecombinant PD-1 fusion protein from transfected CHO cells at 1-2 μg/mlin PBS, 100 μl/wells incubated 4° C. overnight then blocked with 200μl/well of 5% fetal bovine serum in PBS/Tween (0.05%). Dilutions of serafrom PD-1-immunized mice were added to each well and incubated for 1-2hours at ambient temperature. The plates were washed with PBS/Tween andthen incubated with a goat-anti-human IgG polyclonal antibody conjugatedwith horseradish peroxidase (HRP) for 1 hour at room temperature. Afterwashing, the plates were developed with ABTS substrate (Sigma, A-1888,0.22 mg/ml) and analyzed by spectrophotometer at OD 415-495. Mice thatdeveloped the highest titers of anti-PD-1 antibodies were used forfusions. Fusions were performed as described below and hybridomasupernatants were tested tor anti-PD-1 activity by ELISA.

Generation of Hybridomas Producing Human Monoclonal Antibodies to PD-1:

The mouse splenocytes, isolated from the HuMab or KM mice, were fused toa mouse myeloma cell line either using PEG based upon standard protocolsor electric field based electrofusion using a Cyto Pulse large chambercell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie,Md.). The resulting hybridomas were then screened for the production ofantigen-specific antibodies. Single cell suspensions of splenocytes fromimmunized mice were fused to one-fourth the member of SP2/0 nonsecretingmouse myeloma cells (ATCC, CRL 1581) with 50% PEG (Sigma). Cells wereplated at approximately 1×10⁵/well in flat bottom microliter plate,followed by about two week incubation in selective medium containing 10%fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium,3-5% origen (IGEN) in DMEM (Mediatech, CRL 10013, with high glucose,L-glutamine and sodium pyruvate) plus 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 mg/ml gentamycin and 1×HAT (Sigma, CRL P-7185).After 1-2 weeks, cells were cultured in medium in which the HAT wasreplaced with HT. Individual wells were then screened by ELISA(described above) for human anti-PD-1 monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored usually after10-14 days. The antibody-secreting hybridomas were replated, screenedagain and, if still positive for human IgG, anti-PD-1 monoclonalantibodies were subcloned at least twice by limiting dilution. Thestable subclones were then cultured in vitro to generate small amountsof antibody in tissue culture medium for further characterization.

Hybridoma clones 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 were selectedfor further analysis.

Example 2 Structural Characterization of Human Monoclonal Antibodies17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4

The cDNA sequences encoding the heavy and light chain variable regionsof the 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 monoclonal antibodies wereobtained from the 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 hybridomas,respectively, using standard PCR techniques and were sequenced usingstandard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of 17D8 are shown in FIG. 1A and in SEQ ID NO: 57 and 1,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 17D8 are shown in FIG. 1B and in SEQ ID NO: 64 and 8,respectively.

Comparison of the 17D8 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 17D8 heavy chain utilizes a VH segment from human germline VH 3-33,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 17D8 VH sequence to the germline VH 3-33 sequenceis shown in FIG. 8. Further analysis of the 17D8 VH sequence using theKabat system of CDR region determination led to the delineation of theheavy chain CDR1, CDR2 and CD3 regions as shown in FIGS. 1A and 8, andin SEQ ID NOs: 15, 22 and 29, respectively.

Comparison of the 17D8 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 17D8 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 4. The alignment of the 17D8 VLsequence to the germline VK L6 sequence is shown in FIG. 9. Furtheranalysis of the 17D8 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 1B and 9, and in SEQ ID NOs: 36, 43 and50, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 2D3 are shown in FIG. 2A and in SEQ ID NO: 58 and 2,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 2D3 are shown in FIG. 2B and in SEQ ID NO: 65 and 9,respectively.

Comparison of the 2D3 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 2D3 heavy chain utilizes a VH segment from human germline VH 3-33, aD segment from human germline 7-27, and a JH segment from human germlineJH 4b. The alignment of the 2D3 VH sequence to the germline VH 3-33sequence is shown in FIG. 8. Further analysis of the 2D3 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 2A and 8, and in SEQ ID NOs: 16, 23 and 30, respectively.

Comparison of the 2D3 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 2D3 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 4. The alignment of the 2D3 VLsequence to the germline VK L6 sequence is shown in FIG. 9. Furtheranalysis of the 2D3 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 2B and 9, and in SEQ ID NOs: 37, 44 and51, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 4H1 are shown in FIG. 3A and in SEQ ID NO: 59 and 3,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 4H1 are shown in FIG. 3B and in SEQ ID NO: 66 and 10,respectively.

Comparison of the 4H1 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 4H1 heavy chain utilizes a VH segment from human germline VH 3-33,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 4H1 VH sequence to the germline VH 3-33 sequence isshown in FIG. 8. Further analysis of the 4H1 VH sequence using the Kabatsystem of CDR region determination led to the delineation of the heavychain CDR1, CDR2 and CD3 regions as shown in FIGS. 3A and 8, and in SEQID NOs: 17, 24 and 31, respectively.

Comparison of the 4H1 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 4H1 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 1. The alignment of the 4H1 VLsequence to the germline VK L6 sequence is shown in FIG. 10. Furtheranalysis of the 4H1 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3B and 10, and in SEQ ID NOs: 38, 45 and52, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 5C4 are shown in FIG. 4A and in SEQ ID NO: 60 and 4,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 5C4 are shown in FIG. 4B and in SEQ ID NO: 67 and 11,respectively.

Comparison of the 5C4 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 5C4 heavy chain utilizes a VH segment from human germline VH 3-33,an undetermined D segment, and a JH segment from human germline JH 4b.The alignment of the 5C4 VH sequence to the germline VH 3-33 sequence isshown in FIG. 8. Further analysis of the 5C4 VH sequence using the Kabatsystem of CDR region determination led to the delineation of the heavychain CDR1, CDR2 and CD3 regions as shown in FIGS. 4A and 8, and in SEQID NOs: 18, 25 and 32, respectively.

Comparison of the 5C4 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 5C4 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 1. The alignment of the 5C4 VLsequence to the germline VK L6 sequence is shown in FIG. 10. Furtheranalysis of the 5C4 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 4B and 10, and in SEQ ID NOs: 39, 46 and53, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 4A11 are shown in FIG. 5A and in SEQ ID NO: 61 and 5,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 4A11 are shown in FIG. 5B and in SEQ ID NO: 68 and 12,respectively.

Comparison of the 4A11 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 4A11 heavy chain utilizes a VH segment from human germline VH 4-39,a D segment from human germline 3-9, and a JH segment from humangermline JH 4b. The alignment of the 4A11 VH sequence to the germline VH4-39 sequence is shown in FIG. 11. Further analysis of the 4A11 VHsequence using the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 5A and 11, and in SEQ ID NOs: 19, 26 and 33, respectively.

Comparison of the 4A11 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 4A11 light chain utilizes a VL segment from human germline VK L15and a JK segment from human germline JK 1. The alignment of the 4A11 VLsequence to the germline VK L6 sequence is shown in FIG. 12. Furtheranalysis of the 4A11 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 5B and 12, and in SEQ ID NOs: 40, 47 and54, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 7D3 are shown in FIG. 7A and in SEQ ID NO: 62 and 6,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 7D3 are shown in FIG. 7B and in SEQ ID NO: 69 and 13,respectively.

Comparison of the 7D3 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 7D3 heavy chain utilizes a VH segment from human germline VH 3-33, ahuman germline 7-27 D segment, and a JH segment from human germline JH4b. The alignment of the 7D3 VH sequence to the germline VH 3-33sequence is shown in FIG. 8. Further analysis of the 7D3 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 6A and 8, and in SEQ ID NOs: 20, 27 and 34, respectively.

Comparison of the 7D3 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 7D3 light chain utilizes a VL segment from human germline VK L6 anda JK segment from human germline JK 4. The alignment of the 7D3 VLsequence to the germline VK L6 sequence is shown in FIG. 9. Furtheranalysis of the 7D3 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 6B and 9, and in SEQ ID NOs: 41, 48 and55, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 5F4 are shown in FIG. 7A and in SEQ ID NO: 63 and 7,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 5F4 are shown is FIG. 7B and in SEQ ID NO: 70 and 14,respectively.

Comparison of the 5F4 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 5F4 heavy chain utilizes a VH segment from human germline VH 4-39, aD segment from human germline 3-9, and a JH segment from human germlineJH 4b. The alignment of the 5F4 VH sequence to the germline VH 4-39sequence is shown in FIG. 11. Further analysis of the 5F4 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 7A and 11, and in SEQ ID NOs: 21, 28 and 35, respectively.

Comparison of the 5F4 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 5F4 light chain utilizes a VL segment from human germline VK L15 anda JK segment from human germline JK 1. The alignment of the 5F4 VLsequence to the germline VK L6 sequence is shown in FIG. 12. Furtheranalysis of the 5F4 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 7B and 12, and in SEQ ID NOs: 42, 49 and56, respectively.

Example 2 Characterization of Binding Specificity and Binding Kineticsof Anti-PD-1 Human Monoclonal Antibodies

In this example, binding affinity and binding kinetics of anti-PD-1antibodies were examined by Biacore analysis. Binding specificity, andcross-competition were examined by flow cytometry.

Binding Affinity and Kinetics

Anti-PD-1 antibodies were characterized for affinities and bindingkinetics by Biacore analysis (Biacore AB, Uppsala, Sweden). Purifiedrecombinant human PD-1 fusion protein was covalently linked to a CM5chip (carboxy methyl dextran coated chip) via primary amines, usingstandard amine coupling chemistry and kit provided by Biacore. Bindingwas measured by flowing the antibodies in HBS EP buffer (provided byBiacore AB) at a concentration of 267 nM at a low rate of 50 μl/min. Theantigen-antibody association kinetics was followed for 3 minutes and thedissociation kinetics was followed for 7 minutes. The association anddissociation curves were fit to a 1:1 Langmuir binding model usingBIAevaluation software (Biacore AB). To minimize the effects of avidityin the estimation of the binding constants, only the initial segment ofdata corresponding to association and dissociation phases were used forfitting. The K_(D), k_(on) and k_(off) values that were determined areshown in Table 2.

TABLE 2 Biacore binding data for PD-1 human monoclonal antibodies.Affinity K_(D) × On rate k_(on) × Off rate k_(off) × Sample # Sample ID10⁻⁹ (M) 10⁵ (1/Ms) 10⁻⁴ 1/s 1 17D8 0.16 2.56 0.45 2 2D3 1.20 3.77 4.523 4H1 5.46 3.15 1.72 4 5C4 0.73 4.32 3.15 5 4A11 0.13 0.76 0.099 6 7D32.49 18.2 4.54 7 5F4 2.91 8.74 2.54

Binding Specificity by Flow Cytometry

Chinese hamster ovary (CHO) cell lines that express recombinant humanPD-1 at the cell surface were developed and used to determine thespecificity of PD-1 human monoclonal antibodies by flow cytometry. CHOcells were transfected with expression plasmids containing full lengthcDNA encoding transmembrane forms of PD-1. Binding of the 5C4 and 4H1anti-PD-1 human monoclonal antibodies was assessed by incubating thetransfected cells with the anti-PD-1 human monoclonal antibodies at aconcentration of 20 μg/ml. The cells were washed and binding wasdetected with a FITC-labeled anti-human IgG Ab. Flow cytometric analyseswere performed using a FACScan flow cytometry (Becton Dickinson, SanJose, Calif.). The results are depleted in FIGS. 13A (5C4) and 13B(4H1). The anti-PD-1 human monoclonal antibodies bound to the CHO cellstransfected with PD-1 but not to CHO cells that were not transfectedwith human PD-1. These data demonstrate the specificity of anti-PD-1human monoclonal antibodies for PD-1.

Binding Specificity by ELISA Against Other CD28 Family Members

A comparison of the binding of anti-PD-1 antibodies to CD28 familymembers was performed by standard ELISA using four different CD28 familymembers to examine the specificity of binding for PD-1.

Fusion proteins of CD28 family members, ICOS, CTLA-4 and CD28 (R&DBiosystems) were tested for binding against the anti-PD-1 humanmonoclonal antibodies 17D8, 2D3, 4H1, 5C4, and 4A11. Standard ELISAprocedures were performed. The anti-PD-1 human monoclonal antibodieswere added at a concentration of 20 μg/ml. Goat-anti-human IgG (kappachain-specific) polyclonal antibody conjugated with horseradishperoxidase (HRP) was used as secondary antibody. The results are shownin FIG. 14. Each of the anti-PD-1 human monoclonal antibodies 17D8, 2D3,4H1, 5C4, 4A11, 7D3 and 5F4 bound with high specificity to PD-1, but notto the other CD28 family members.

Example 4 Characterization of Anti-PD-1 Antibody Binding to PD-1Expressed on the Surface of Human and Monkey Cells

Anti-PD-1 antibodies were tested for binding to cells expressing PD-1 ontheir cell surface by flow cytometry.

Activated human T-cells, monkey peripheral blood mononuclear cells(PBMC), and CHO cells transfected with PD-1 were each tested forantibody binding. Human T cells and cynomolgous PBMC were activated byanti-PD-1 antibody to induce PD-1 expression on T cells prior to bindingwith a human anti-PD-1 monoclonal antibody. Binding of the 5C4 and 4H1anti-PD-1 human monoclonal antibodies was assessed by incubating thetransfected cells with either IgG1 or IgG4 forms of the anti-PD-1 humanmonoclonal antibodies at different concentrations. The cells were washedand binding was detected with a FITC-labeled anti-human IgG Ab. Flowcytometric analyses were performed using a FACScan flow cytometry(Becton Dickinson, San Jose, Calif.). The results are shown in FIGS. 15A(activated human T cells), 15B (cynomolgous monkey PBMC) and 15C(PD-1-transfected CHO cells). The anti-PD-1 monoclonal antibodies 5C4and 4H1 bound to activated human T cells, activated monkey PBMCs, andCHO cells transfected with human PD-1, as measured by the meanfluorescent intensity (MFI) of staining. These data demonstrate that theanti-PD-1 HuMAbs bind to both human and cynomolgous monkey cell surfacePD-1.

Example 5 Effect of Human Anti-PD-1 Antibodies on Cell Proliferation andCytokine Production in a Mixed Lymphocyte Reaction

A mixed lymphocyte reaction was employed to demonstrate the effect ofblocking the PD-1 pathway to Lymphocyte effector cells. T cells in theassay were tested for proliferation, IFN-gamma secretion and IL-2secretion in the presence or absence of an anti-PD-1 HuMAb antibody.

Human T-cells were purified from PBMC using a human CD4+ T cellenrichment column (R&D systems). Each culture contained 10⁵ purifiedT-cells and 10⁴ allogeneic dendritic cells in a total volume of 200 μl.Anti-PD-1 monoclonal antibody 5C4, 4H1, 17D8, 2D3 or a Fab fragmentportion of 5C4 was added to each culture at different antibodyconcentrations. Either no antibody or an isotype control antibody wasused as a negative control. The cells were cultured for 5 days at 37° C.After day 5, 100 μl of medium was taken from each culture for cytokinemeasurement The levels of IFN-gamma and other cytokines were measuredusing OptEIA ELISA kits (BD Biosciences). The cells were labeled with³H-thymidine, cultured for another 18 hours, and analyzed for cellproliferation. The results are shown in FIGS. 16A (T cellproliferation), 16B (IFN-γ secretion) and 16C (IL-2 secretion). Theanti-PD-1 human monoclonal antibodies promoted T-cell proliferation,IFN-gamma secretion and IL-2 secretion in a concentration dependentmanner. The 5C4-Fab fragment also promoted T-cell proliferation,IFN-gamma secretion and IL-2 secretion in a concentration dependentmanner. In contrast, cultures containing the isotype control antibodydid not show an increase in T cell proliferation, IFN-gamma or IL-2secretion.

Example 6 Blocking of Ligand Binding to PD-1 by Human Anti-PD-1Antibodies

Anti-PD-1 HuMAbs were tested for the ability to block binding of theligands PD-L1 and PD-L2 to PD-1 expressed on transfected CHO cells byusing a flow cytometry assay.

PD-1 expressing CHO cells were suspended in FACS buffer (PBS with 4%fetal calf serum). Various concentrations of the anti-PD-1 HuMAbs 5C4and 4H1 were added to the cell suspension and incubated at 4° C. for 30minutes. Unbound antibody was washed off and either FTTC-labeled PD-L1fusion protein or FITC-labeled PD-L2 fusion protein was added into thetubes and incubated at 4° C. for 30 minutes. Flow cytometric analyseswere performed using a FACScan flowcytometer (Becton Dickinson, SanJose, Calif.). The results are depleted in FIGS. 1A (blocking of PD-L1)and 1B (blocking of PD-L2). The anti-PD-1 monoclonal antibodies 5C4 and4H1 blocked binding of PD-L1 and PD-L2 to CHO cells transfected withhuman PD-1, as measured by the mean fluorescent intensity (MFI) ofstaining. These data demonstrate that the anti-PD-1 HuMAbs block bindingof ligand (both PD-L1 and PD-L2) to cell surface PD-1.

Example 7 Effect of Human Anti-PD-1 Antibodies on the Release ofCytokines in Human Blood

The anti-PD-1 HuMAbs were mixed with fresh human whole blood in order todetermine whether the anti-PD-1 HuMAbs alone simulated the release ofcertain cytokines from human blood cells.

500 μl of heparinized-fresh human whole blood, was added into each well.Either 10 μg or 100 μg of an anti-PD-1 HuMAb (4H1 or 5C4, the lattereither as an IgG1 or IgG4 isotype) was added to each well. Some wellswere incubated with anti-CD3 antibody as a positive control, or a humanIgG1 or human IgG4 antibody as isotype-matched negative controls. Thecells were incubated at 37° C. for either 6 or 24 hours. The cells werespun down and the plasma was collected for measurement of the cytokinesIFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-10 and IL-12 using a cytokinecytometric bead array assay (BD Biosciences). The concentration of eachcytokine (pg/ml) is shown in Tables 3a, with a 6 hour incubation, and3b, with a 24 hour incubation, below. The results show that treatmentwith the human anti-PD-1 antibodies 5C4 and 4H1 alone did not stimulatehuman blood cells to release any of the cytokines IFN-gamma, TNF-alpha,IL-2, IL-4, IL-6, IL-10 and IL-12.

TABLE 3a Cytokine production following 6 hour incubation IFN- TNF- gammaalpha IL-10 IL-6 IL-4 IL-2 Ab (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)(pg/ml) No Ab 12.3 2 3 5 3.6 1.9 10 mg/ml 5000 530 82.6 510.4 37.2 467.9anti-CD3 100 mg/ml 5000 571 91.3 530 43.9 551.5 anti-CD3 10 mg/ml 7 1.82.8 4.4 2.6 1.5 hIgG1 100 mg/ml 0 2.2 2.7 6 2.6 1.4 hIgG1 10 mg/ml 5.41.4 2.5 4.5 2.1 1.3 hIgG4 100 mg/ml 6.4 2.3 3 32.6 2.9 1.4 hIgG4 10mg/ml 6.2 1.8 2.4 4.1 2.8 1.6 4H1 100 mg/ml 11.8 2 2.6 3.5 2.6 1.7 4H110 mg/ml 4.2 1.6 2.3 3.9 2.5 1.3 5C4 IgG1 100 mg/ml 0 1.4 2.2 3.6 2.11.2 5C4 IgG1 10 mg/ml 8.3 2.5 1.9 4.8 1.6 1.5 5C4 IgG4 100 mg/ml 3.6 1.72.4 3.9 2.3 1.5 5C4 IgG4

TABLE 3b Cytokine production following 24 hour incubation IFN- TNF-gamma alpha IL-10 IL-6 IL-4 IL-2 Ab (pg/ml) (pg/ml) (pg/ml) (pg/ml)(pg/ml) (pg/ml) No Ab 11.2 2 6.1 5.9 2.6 1.7 10 mg/ml 5000 565.9 4325000 64.5 1265.3 anti-CD3 100 mg/ml 5000 535 461 5000 73.8 1334.9anti-CD3 10 mg/ml 0 0 0 0 0 0 hIgG1 100 mg/ml 11.5 1.7 7.9 60.8 2.9 1.5hIgG1 10 mg/ml 24.6 3.1 8.3 63.4 3.1 2.3 hIgG4 100 mg/ml 11.2 1.8 8 27.73.1 2.4 hIgG4 10 mg/ml 27.3 2.9 8 13.9 5.3 2.6 4H1 100 mg/ml 17.5 2.54.4 7 4 2.1 4H1 10 mg/ml 9.1 2 7.6 68.5 3.5 1.8 5C4 IgG1 100 mg/ml 12.91.9 6.1 25.3 2.9 1.7 5C4 IgG1 10 mg/ml 14 1.9 4.4 3.3 2.6 1.9 5C4 IgG4100 mg/ml 0 0 0 0 0 0 5C4 IgG4

Example 8 Effect of Anti-PD-1 Antibodies on the Apoptosis of T-Cells

The effect of anti-PD-1 antibodies on the induction of apoptosis ofT-cells was measured using an annexin V staining test.

T cells were cultured in a mixed lymphocyte reaction, as described abovein Example 5. The anti-PD-1 antibody 5C4 was added to the tube at aconcentration of 25 μg/ml. A non-specific antibody was used as acontrol. Annexin V and propidium iodide were added according to standardprotocol (BD Biosciences). The mixture was incubated for 15 minutes inthe dark at room temperature and then analyzed using a FACScanflowcytometer (Becton Dickinson, San Jose, Calif.). The results areshown in FIG. 18. The anti-PD-1 antibody 5C4 did not have an effect onT-cell apoptosis.

Example 9 Effect of Anti-PD-1 Antibodies on Cytokine Secretion byViral-Stimulated PBMC Cells from a Virus Positive Donor

In this example, peripheral blood mononuclear cells (PBMC) from a donorpositive for CMV were isolated and exposed to a CMV lysate in thepresence or absence of anti-PD-1 antibodies to examine the effect of theantibodies on cytokine secretion simulated by antigen.

2×10⁵ human PMBCs from a CMV positive donor were cultured in a totalvolume of 200 μl and added into each well along with a lysate ofCMV-injected cells. The anti-PD-1 HuMAb 5C4 was added to each well invarious concentrations for 4 days. After day 4, 100 μl of medium wastaken from each culture for cytokine measurement. The level of IFN-gammawas measured using OptEIA-ELISA bits (BD Biosciences). The cells werelabeled with ³H-thymidine, cultured for another 18 hours, and analyzedfor cell proliferation. The cell proliferation was analyzed using theCell Titer-Glo reagent (Promega). The results are shown in FIG. 19. Theanti-PD-1 HuMab 5C4 increased IFN gamma secretion in a concentrationdependent manner. These results shows that anti-PD-1 HuMAbs canstimulate IFN-gamma release in a memory T cell response from PBMC cellspreviously stimulated against an antigen.

Example 8 Effect of Anti-PD-1 Antibody on Secondary Antibody Response toAntigen

Mice were immunized and rechallenged with a T1-antigen (DNP-Ficoll) andalso treated with a rat anti-mouse-PD-1 antibody, or a control antibodyto examine the effect of the anti-PD-1 antibody on antibody titers.

Female C57BL6 mice were divided into two groups, with 6 mice/group. Onegroup was treated with a control rat IgG and the other with a ratanti-mouse PD-1 antibody. The mice were immunized with 5 μg ofDNP-Ficoll (a T1-antigen) in 50 μl CFA by i.p. at day 0. Either thecontrol rat IgG antibody or the rat-mPD-1 antibody (200 μg/mouse) wasgiven by i.p. at days −1, 0 and 2. Four weeks later, mice wererechallenged with 5 μg of DNP-Ficoll in 50 μl IFA by i.p. at day 0. Ratanti-mPD-1 antibody or control antibody (200 μg/mouse) was given by i.p.at days 0 and 1. Antibody titers were measured by standard ELISA assayat day 7 following the boost. The results are shown in Table 4 below. Inthe mice treated with the anti-mPD-1 antibody, both IgM and IgG3isotypes showed the greatest increase in titer following challenge withthe T1-antigen, as compared to mice treated with a control antibody.These results demonstrate that anti-PD-1 treatment can increase antibodytiters in response to T1-antigen.

TABLE 4 Murine secondary response following treatment with anti-PD-1antibody Antibody Rat anti-mouse PD- Isotype Control group 1 antibody Pvalue IgM 606 1200 0.026 IgG 9 15.55 0.18 IgG1 1.2 1.1 0.83 IgG2b 5.059.26 0.18 IgG3 21.9 81.2 0.03 * Results shown are average concentrationof antibody isotype (μg/ml)

Example 11 Treatment of In Vivo Tumor Model Using Anti-OD-1 Antibodies

Mice implanted with a cancerous tumor were treated in vivo withanti-PD-1 antibodies to examine the in vivo effect of the antibodies ontumor growth. As a positive control, an anti-CTLA-4 antibody was used,since such antibodies have been shown to inhibit tumor growth in vivo.

In this experiment, the anti-PD-1 antibody used was a chimeric ratanti-mouse-PD-1 antibody generated using well known laboratorytechniques. To generate the rat anti-mouse PD-1 antibody, rats wereimmunized with mouse cells transfected to express a recombinant mousePD-1 fusion protein (R&D Systems Catalog No. 1021-PD) and monoclonalantibodies were screened for binding to mouse PD-1 antigen by ELISAassay. The rat anti-PD-1 antibody V regions were then recombinantlylinked to a murine IgG1 constant region using standard molecular biologytechniques and rescreened for binding to mouse PD-1 by ELISA and FACS.The chimeric rat anti-moose-PD-1 antibody used herein is referred to as4H2.

For the tumor studies, female AJ mice between 6-8 weeks of age (HarlanLaboratories) were randomized by weight into 6 groups. The mice wereimplanted subcutaneously in the right flank with 2×10⁶ SA1/Nfibrosarcoma cells dissolved in 200 μl of DMEM media on day 0. The micewere treated with PBS vehicle, or antibodies at 10 mg/kg. The animalswere dosed by intraperitoneal injection with approximately 200 μl of PBScontaining antibody or vehicle on days 1, 4, 8 and 11. Each groupcontained 10 animals and the groups consisted of: (i) a vehicle group,(ii) control mouse IgG, (iii) control hamster IgG, (iv) hamsteranti-mouse CTLA-4 antibody and (v) the chimeric anti-PD-1 antibody 4H2.The mice were monitored twice weekly for tumor growth for approximately6 weeks. Using an electronic caliper, the tumors were measured threedimensionally (height×width×length) and tumor volume was calculated.Mice were euthanized when the tumors reached tumor end point (1500 mm³)or show greater than 15% weight loss. The results are shown in FIG. 20.The anti-PD-1 antibody extended the mean time to reaching the tumor endpoint volume (1500 mm³) from ˜25 days in the control groups to ˜40 days.Thus, treatment with an anti-PD-1 antibody has a direct in vivoinhibitory effect on tumor growth.

Example 12 Generation of Chimeric (Rat-Mouse) Anti-PD-1 Antibody 4H2

Rat monoclonal antibody against mouse PD-1 antibodies (rat anti-mPD-1)were generated from rats immunized with mPD-1-hFc fusion protein usingstandard hybridoma production methods (see Kohler and Milstein (1975)Nature 256:495; and Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.).Eight hybridomas were subcloned, and antibodies were isolated andscreened for their ability to block mouse PD-L2 (mPD-L2) binding tomPD-1. Several anti-mPD-1 antibodies capable of blocking mPD-L2 bindingto mPD-1 were identified (see, e.g., activity of 4H2, FIG. 41) and thebinding affinity of several of these antibodies to mPD-1-Fc fusionprotein was determined by ELISA (FIG. 42).

Antibody 4H2.B3 was further characterized, which is referred tointerchangeably herein as “4H2.” CHO cells expressing mouse PD-1 wereconstructed and incubated with 4H2 anti-mPD-1 antibody at aconcentration ranging from 200 μg/ml to 0.012 μg/ml to determine thebinding affinity of 4H2 to PD-1. Binding of anti-mPD-1 antibody to thePD-1 expressing CHO cells was detected by incubating withdonkey-anti-rat IgG, FITC conjugated and measured by FACS. The anti-PD-1antibody had an EC₅₀ (50% effective concentration) of about 0.38 μg(FIG. 43) and a K_(D) of 4.7×10⁻⁹ M. To examine the inhibition of PD-L1binding to PD-1, the same assay was performed except that the cells werealso incubated with 0.16 μg mPD-L1-hFc fusion protein, then binding ofPD-L1 to the PD-1 expressing CHO cells was detected by incubating withgoat-anti-human IgG (Fc specific), FITC conjugated and measuring bindingsignal by FACS (MFI, mean fluorescence intensity). The anti-mPD-1antibody had an EC₅₀ of about 0.72 μg (FIG. 44).

For use in the mouse tumor models, the 4H2 rat anti-mPD-1 needed to bemodified so the mouse immune system would not neutralize theimmunotherapeutic antibody (i.e., so the antibody would have betterpharmacokinetics) and to avoid antibody-dependent cellular cytotoxicity(ADCC) by reducing Fc receptor interactions (i.e., so blockade byanti-PD-1 could be evaluated with being compromised by ADCC effects).The original rat anti-mPD-1 antibody, 4H2, was determined to be a ratIgG2a isotype. Hence, the Fc-portion of the 4H2 antibody was replacedwith an Fc-portion from a mouse IgG1 isotype. Using the assay describedabove, the binding affinity of the rat-mouse chimeric 4H2 to mPD-1 wasfound to be comparable to the rat 4H2.B3 anti-mPD-1 antibody (FIG. 45).Similarly, inhibition of PD-L1 binding to PD-1 was comparable for bothantibodies (FIG. 46). Thus, the rat-mouse chimeric 4H2 anti-mPD-1antibody was used to examine the therapeutic efficacy of anti-PD-1 incombination with anti-CTLA-4.

Example 13 In Vivo Efficacy of Combination Therapy (Anti-CTLA-4 andAnti-PD-1 Antibodies) on Tumor Establishment and Growth

MC38 colorectal cancer cells (PD-L1⁻) (available from Dr. N. Restifo,National Cancer Institute, Bethesda, Md.; or Jeffrey Schlom, NationalInstitutes of Health, Bethesda, Md.) were implanted in C57BL/6 mice(2×10⁶ cells/mouse). On day 0 (i.e., the day the MC38 cells wereimplanted in the mice), each of four groups of 10 mice each was injectedintraperitoneally (IP) with one of the following: (1) mouse IgG(control), (2) anti-CTLA-4 monoclonal antibody 9D9 (mouse anti-mouseCTLA-4, obtained from J. Allison, Memorial Sloan-Kettering CancerCenter, New York, N.Y.), (3) anti-PD-1 monoclonal antibody 4H2 (chimericantibody in which a rat anti-mouse PD-1 was modified with a mouse Fcregion, as described in Example 6), or (4) anti-CTLA-4 antibody 9D9 andanti-PD-1 antibody 4H2. Antibody injections were then furtheradministered on days 3, 6 and 10. The single antibody treatments weredosed at 10 mg/kg, and the combination of anti-CTLA-4 antibody andanti-PD-1 antibody was dosed at 5 mg/kg of each antibody (i.e., 10 mg/kgof total antibody). Using an electronic caliper, the tumors weremeasured three dimensionally (height×width×length) and tumor volume wascalculated. Mice were euthanized when the tumors reached a designatedtumor end-point. The results are shown in Table 5 and FIG. 21.

TABLE 5 Percentage of Tumor-Free Mice Following Anti-PD-1 and/orAnti-CTLA-4 Treatment Treatment Total mice studied Tumor-free mice (%)mIgG1 10 0 anti-CTLA-4 10 1 (10) anti-PD-1 10 3 (30) anti-CTLA-4 +anti-PD-1 10 6 (60)

Eight mice in the IgG group reached the tumor end-point by about day 30and two mice (86066 and 87260) in the IgG group had ulcerated tumors(FIG. 21A). In the anti-CTLA-4 antibody alone group, seven mice reachedthe tumor end-point by about day 60, one mouse had an ulcerated tumor(84952), one mouse had a tumor with a volume of less than 1500 mm³(85246), and one mouse was tumor-free (86057) (FIG. 21B). In theanti-PD-1 antibody alone group, six mice reached the tumor end-point byabout day 60, one mouse had an ulcerated tumor (86055), and three micewere tumor-free (84955, 85239 and 86750) (FIG. 21C). In the anti-CTLA-4antibody and anti-PD-1 antibody combination group, four mice reached thetumor end-point by about Day 40, and six mice were tumor-free (84596,85240, 86056, 86071, 86082 and 86761) (FIG. 21D).

FIG. 22 shows that the mean tumor volume measured at day 21 was about2955 mm³ for the IgG control group; about 655 mm³ for the CTLA-4antibody alone group, about 510 mm³ for the PD-1 antibody alone group,and about 280 mm³ for the anti-CTLA-4 antibody and anti-PD-1 antibodycombination group. FIG. 23 shows that the median tumor volume measuredat day 21 was about 2715 mm³ for the IgG group; about 625 mm³ for theCTLA-4 antibody alone group; about 525 mm³ for the PD-1 antibody alonegroup; and about 10 mm³ for the CTLA-4 antibody and PD-1 antibodycombination group (and down to 0 mm³ by day 32).

This study indicates that, in a murine tumor model, CTLA-4 antibodytreatment alone and PD-1 antibody treatment alone have a modest effecton tumor growth, and that the combination treatment of CTLA-4 antibodyand PD-1 antibody has a significantly greater effect on tumor growth. Itis interesting to note that the combination treatment with CTLA-4antibody and PD-1 antibody had a more significant effect on tumor growthat a dose of 5 mg/kg of each antibody as compared to the effect ofeither antibody alone when each is administered at a higher dose of 10mg/kg.

Example 14 In Vivo Efficacy of Combination Therapy (Anti-CTLA-4 andanti-PD-1 Antibodies) on Established Tumor Growth

MC38 colorectal cancer cells (PD-L1⁻) were implanted in C57BL/6 mice(2×10⁶ cells/mouse) for a time sufficient (about 6 to 7 days) to permitthe formation of tumors. On day 6 post-implantation (day −1), tumormeasurements were taken and mice were randomized based on mean tumorvolume (about 250 mm³) into 11 groups for subsequent antibody therapy.At day 0 (i.e., one week after the MC38 cells were implanted), mice wereinjected IP with (1) mouse IgG (control), (2) anti-CTLA-4 monoclonalantibody 9D9, (3) anti-PD-1 monoclonal antibody 4H2, or (4) anti-CTLA-4monoclonal antibody 9D9 and anti-PD-1 antibody monoclonal antibody 4H2,at a concentration of 10 mg/kg per mouse. Antibody injections were alsoadministered on days 3, 6 and 10. The monoclonal antibody compositionsused had low levels of endotoxin and did not significantly aggregate.Using an electronic caliper, the tumors were measured threedimensionally (height×width×length) and tumor volume was calculated.Tumor measurements were taken on day 0 (tumors at the beginning oftreatment had a volume of about 125 mm³), and on days 3, 6, 10, 13, 17and 20 post-antibody injection. Mice were euthanized when the tumorsreached a designated tumor end-point (a particular tumor volume such as1500 mm³ and/or when the mice showed greater than about 15% weightloss).

All eleven mice in the IgG group reached the tumor end-point by aboutday 17 (FIG. 24A). In the anti-CTLA-4 antibody alone group, seven ofeleven mice reached the tumor end-point by about day 12 (FIG. 24B). Inthe anti-PD-1 antibody alone group, four mice reached the tumorend-point by about day 13 and two mice were tumor-free (FIG. 24C). Inthe anti-CTLA-4 antibody and anti-PD-1 antibody combination group, onemouse reached the tumor end-point by about day 17, one mouse reached thetumor end-point by about day 45 and nine mice were tumor-free on day 45(FIG. 24D).

FIG. 25 shows that the mean tumor volume measured at day 10 was about1485 mm³ for the IgG control group; about 1010 mm³ for the CTLA-4antibody alone group; about 695 mm³ for the PD-1 antibody alone group;and about 80 mm³ for the anti-CTLA-4 antibody and anti-PD-1 antibodycombination group. FIG. 26 shows that the median tumor volume measuredat day 10 was about 1365 mm³ for the IgG group; about 1060 mm³ for theanti-CTLA-4 antibody alone group; about 480 mm³ for the anti-PD-1antibody alone group; and about 15 mm³ for the anti-CTLA-4 antibody andanti-PD-1 antibody combination group (which was down to 0 mm³ by day17).

This study indicates that, in a murine tumor model, treatment with thecombination of CTLA-4 antibody and PD-1 antibody has a significantlygreater effect on tumor growth than either antibody alone, even when atumor is already well established.

Example 15 Dose Titration of Combination Therapy (Anti-CTLA-4 andAnti-PD-1 Antibodies) on Established Tumor Growth

MC38 colorectal cancer cells (PD-L1⁻) were implanted in C57BL/6 mice(2×10⁶ cells/mouse) for a time sufficient (about 6 to 7 days) to permitthe formation of tumors as described in Example 3. Groups of 10 micewere injected IP at days 0, 3, 6 and 10 as follows: Group (A) mouse IgG(control, 20 mg/kg), Group (B) anti-PD-1 monoclonal antibody 4H2 (10mg/kg) and mouse IgG (10 mg/kg), Group (C) anti-CTLA-4 monoclonalantibody 9D9 (10 mg/kg) and mouse IgG (10 mg/kg), Group (D) anti-CTLA-4monoclonal antibody 9D9 (10 mg/kg) and anti-PD-1 antibody monoclonalantibody 4H2 (10 mg/kg), Group (E) anti-CTLA-4 monoclonal antibody 9D9(3 mg/kg) and anti-PD-1 antibody monoclonal antibody 4H2 (3 mg/kg), orGroup (F) anti-CTLA-4 monoclonal antibody 9D9 (1 mg/kg) and anti-PD-1antibody monoclonal antibody 4H2 (1 mg/kg). Using an electronic caliper,the tumors were measured three dimensionally (height×width×length) andtumor volume was calculated. Tumor measurements were taken at thebeginning of treatment (i.e., on day 0 tumors had an average volume ofabout 90 mm³), and on days 3, 6, 10, 13, 17 and 20 post-antibodytreatment. Mice were euthanized when the tumors reached a designatedtumor end-point (a particular tumor volume such as 1500 mm³ and/or whenthe mice showed greater than about 15% weight loss).

FIG. 27A shows that all 10 control mice had reached a tumor end-point.FIG. 27B shows that the group treated with 10 mg/kg anti-PD-1 antibody(Group B) had 6 mice that reached the tumor end-point and 4 mice withtumors having a volume of about 750 mm³ or less. FIG. 27C shows that thegroup treated with 10 mg/kg anti-CTLA-4 antibody (Group C) had 3 micethat reached the tumor end-point and 7 mice with tumors having a volumeof about 1000 mm³ or less. FIG. 27D shows that the group treated with acombination of 10 mg/kg anti-PD-1 antibody with 10 mg/kg anti-CTLA-4antibody (Group D) had 2 mice with tumors having a volume of about 1000mm³ or less, and 8 mice that were tumor free. FIG. 27E shows that thegroup treated with a combination of 3 mg/kg anti-PD-1 antibody with 3mg/kg anti-CTLA-4 antibody (Group B) had one mouse that had reached thetumor end-point, 7 mice with tumors having a volume of about 500 mm³ orless, and 2 mice that were tumor free. FIG. 27F shows that the grouptreated with a combination of 1 mg/kg anti-PD-1 antibody with 1 mg/kganti-CTLA-4 antibody (Group F) had 4 mice that had reached the tumorend-point, 5 mice with tumors having a volume of about 1100 mm³ or less,and one mouse that was tumor free.

FIGS. 27G and 27H show the tumor volumes in mice treated sequentiallywith anti-PD-1 antibody first and anti-CTLA-4 antibody second, and viceversa. The mice of FIG. 27G first received 10 mg/kg anti-CTLA-4 on eachof days 0 and 3, and then received 10 mg/kg anti-PD-1 antibody on eachof days 0 and 10. The mice of FIG. 27H first received 10 mg/kg anti-PD-1antibody on each of days 0 and 3, and then received 10 mg/kg anti-CTLA-4antibody on each of days 6 and 10. For group G at day 27, 8 mice reachedthe tumor end-point, one mouse had a very small tumor (which, after asignificant delay, eventually grew out) and one mouse was tumor free.For group H at day 27,8 mice reached the tumor end-point and 2 weretumor free.

FIG. 28 shows that the mean tumor volume measured at day 10 was about1250 mm³ for the IgG control group; about 470 mm³ for the PD-1 antibodywith the IgG control; about 290 mm³ for the CTLA-4 antibody with the IgGcontrol (measured at day 6); about 40 mm³ for the anti-CTLA-4 antibody(10 mg/kg) and anti-PD-1 antibody (10 mg/kg) combination group; about165 mm³ for the anti-CTLA-4 antibody (3 mg/kg) and anti-PD-1 antibody (3mg/kg) combination group; and about 400 mm³ for the anti-CTLA-4 antibody(1 mg/kg) and anti-PD-1 antibody (1 mg/kg) combination group. FIG. 29shows that the median tumor volume measured at day 13 was about 1680 mm³for the IgG control group; about 400 mm³ for the PD-1 antibody with theIgG control; about 660 mm³ for the CTLA-4 antibody with the IgG control;0 mm³ for the anti-CTLA-4 antibody (10 mg/kg) and anti-PD-1 antibody (10mg/kg) combination group; about 90 mm³ for the anti-CTLA-4 antibody (3mg/kg) and anti-PD-1 antibody (3 mg/kg) combination group; and about 650mm³ for the anti-CTLA-4 antibody (1 mg/kg) and anti-PD-1 antibody (1mg/kg) combination group. For the combination treatment of the anti-PD-1antibody with the anti-CTLA-4 antibody, the number of mice per groupthat were tumor free at day 27 of the study was 8/10 (10 mg/kg), 2/10 (3mg/kg) and 1/10 (1 mg/kg) (data not shown).

This study indicates that, in a murine tumor model, treatment with thecombination of CTLA-4 antibody and PD-1 antibody functions in a dosedependent manner and has a significantly greater effect on tumor growththan both antibodies alone, even at a lower dose and even when a tumoris already well established. Moreover, the antibodies may beadministered sequentially (anti-CTLA-4 antibody first and anti-PD-1antibody second, or vice versa) and the combination is still superior tothe antibody monotherapies.

Example 16 In Vivo Efficacy of Combination Therapy (Anti-CTLA-4 andanti-PD-1 Antibodies) on Fibrosarcoma Establishment and Growth

SA1/N fibrosarcoma cells (PD-L1⁻) (Leach et al. (1996)Science271:1734-1736) were implanted subcutaneously in A/J mice (2×10⁶cells/mouse) on day 0. On days 1, 4, 7 and 11 post-implantation, micewere injected IP as follows: Group (A) PBS alone (referred to as the“vehicle”); Group (B) mouse IgG (control 10 mg/kg per mouse), Group (C)anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse), Group (D)anti-CTLA-4 monoclonal antibody 9D9 (10 mg/kg or 0.2 mg/kg per mouse),and Group (E) anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse) incombination with anti-CTLA-4 monoclonal antibody 9D9 (0.2 mg/kg permouse). The study lasted 41 days and tumor measurements were taken onvarious days throughout the course of the study (see FIG. 29). Tumorvolume was calculated by measuring tumors in three dimensions(height×width×length) using an electronic caliper. Mice were euthanizedwhen the tumors reached a designated tumor end-point—a volume of 1500mm³ and/or an ulcerated tumor.

FIGS. 30A and 30B show that 19 out of the 20 control (9/10 in Group Aand 10/10 in Group B) mice had either reached a tumor end-point or haddeveloped ulcerated tumors. FIG. 30C shows that the group treated with10 mg/kg anti-PD-1 antibody (Group C) had 6 mice that reached a tumorend-point (2 with a volume greater than 1500 mm³ and 4 with an ulceratedtumor) and 4 mice that were tumor free. FIG. 30D shows that the grouptreated with 10 mg/kg anti-CTLA-4 antibody (Group D) had 5 mice thatreached a tumor end-point (2 with a volume greater than 1500 mm³ and 3with an ulcerated tumor), one mouse with a small tumor (volume of about70 mm³) and 4 mice that were tumor free. FIG. 30E shows that the grouptreated with 0.2 mg/kg anti-CTLA-4 antibody (Group E) had 10 mice thatreached a tumor end-point (6 with a volume greater than 1500 mm³ and 4with an ulcerated tumor). FIG. 30F shows that the group treated with acombination of 10 mg/kg anti-PD-1 antibody with 0.2 mg/kg anti-CTLA-4antibody (Group F) had 2 mice that reached a tumor end-point (one with avolume greater than 1500 mm³ and one with an ulcerated tumor) and 8 micethat were tumor free.

FIGS. 31 and 32 show the mean and median tumor volume, respectively,that developed in treated and untreated mice over the course of thisstudy. The tumor growth inhibition in mice treated with theseantibodies, as compared to mice treated with the control antibody mouseIgG, is summarized In Table 6.

TABLE 6 Tumor Growth Inhibition and Tumor Free Mice Following Anti-PD-1and/or Anti-CTLA-4 Treatment Median Median No. of Tumor Vol- Tumor Vol-Tumor ume - mm³ TGI* (%) ume - mm³ TGI (%) Free Mice Group^(†) (Day 15)(Day 15) (Day 19) (Day 19) (Day 41) A 985 — 1140 — 0/10 B 635 — 1060 —0/10 C 465 27 310 71 4/10 D 235 63 90 91 4/10 E 600 6 805 24 0/10 F 33048 90 92 8/10 *TGI = tumor growth inhibition; the median could becalculated only when fewer than 50% of the mice reached the tumor endpoint. ^(†)Groups are as defined in FIG. 30. A = vehicle (PBS); B =mouse IgG; C = anti-PD-1, 10 mg/kg; D = anti-CTLA-4, 10 mg/kg; E =anti-CTLA-4, 0.2 mg/kg; and F = anti-PD-1, 10 mg/kg with anti-CTLA-4,0.2 mg/kg.

These data further indicate that the combination therapy comprisinganti-PD-1 and anti-CTLA-4 antibodies is substantially more effectivethan treatment with either antibody alone. Indeed, the combination isstill more effective than single antibody treatments even when thecombination therapy contains a subtherapeutic dose of anti-CTLA-4antibody. These data also indicate that surprisingly the presence orabsence of PD-L1 on the tumor may have no effect on the efficacy oftreatment with this antibody combination, although the presence of PD-L1may influence the effect of the antibody monotherapies in thatexpression of PD-L1 on the tumor may also lead to inhibition ofanti-tumor T cell responses (see FIG. 40).

Example 17 In Vivo Efficacy and Dose Titration of Combination Therapy(Anti-CTLA-4 and Anti-PD-1 Antibodies) on PD-L1⁻ Fibrosarcoma Growth

SA1/N fibrosarcoma cells (PD-L1⁻) were implanted subcutaneously in A/Jmice (2×10⁶ cells/mouse) on day 0 for a time sufficient (about 7 days)to permit the establishment of a tumor. On days 7, 10, 13 and 16post-implantation, ten groups of 8 mice having an average tumor volumeof 110 mm³ were injected IP as follows: Group (A) PBS alone (referred toas the “vehicle”); Group (B) mouse IgG (control, 10 mg/kg per mouse);Group (C) anti-CTLA-4 monoclonal antibody 9D9 (0.25 mg/kg); Group (D)anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg/kg per mouse); Group (E)anti-CTLA-4 monoclonal antibody 9D9 (5 mg/kg); Group (F) anti-PD-1monoclonal antibody 4H2 (3 mg/kg per mouse); Group (G) anti-PD-1monoclonal antibody 4H2 (10 mg/kg per mouse); Group (H) anti-PD-1monoclonal antibody 4H2 (10 mg/kg per mouse) in combination withanti-CTLA-4 monoclonal antibody 9D9 (0.25 mg/kg per mouse); Group (I)anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse) in combinationwith anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg/kg per mouse); andGroup (J) anti-PD-1 monoclonal antibody 4H2 (3 mg/kg per mouse) incombination with anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg/kg permouse).

On days 10, 13, 16 and 19 post-implantation, two groups of 6 mice havingan average tumor volume of 255 mm³ were injected IP as follows: Group(K) mouse IgG (control, 10 mg/kg per mouse); and Group (L) anti-PD-1monoclonal antibody 4H2 (10 mg/kg per mouse) in combination withanti-CTLA-4 monoclonal antibody 9D9 (1 mg/kg per mouse). The studylasted 51 days and tumor measurements were taken on various daysthroughout the course of the study (see FIGS. 33-38). Tumor volume wascalculated by measuring tumors in three dimensions (height×width×length)using an electronic caliper. Mice were euthanized when the tumorsreached a designated tumor end-point—a volume of 1500 mm³ and/or anulcerated tumor.

FIG. 33 shows the response to immunostimulatory antibody treatment inmice with tumors having an initial volume of about 110 mm³ (i.e., at thetime of the first antibody treatment. FIG. 33A and 33B show that all 16control mice (Groups A and B) reached a tumor end-point (15 with a tumorvolume greater than 1500 MM³ and 1 with an ulcerated tumor). FIGS.33G-33E show that tumor bearing mice respond to treatment withanti-CTLA-4 antibody in a dose-dependent manner (e.g., Group C receiving0.25 mg/kg had 7/8 mice reach the tumor end-point and one mouse with atumor volume less than 200 mm³, whereas Group E receiving 5 mg/kg had6/8 mice reach the tumor end-point and two mice were tumor free). FIGS.33F and 33G show that mice responded about the same regardless of theanti-PD-1 antibody dose (Group F received 3 mg/kg and Group G received10 mg/kg). In contrast, the mice receiving a combination treatment of 10or 3 mg/kg anti-PD-1 antibody with 0.25 or 0.5 mg/kg anti-CTLA-4antibody (Groups H, I and J) showed a significant reduction in tumorgrowth. For example, FIG. 33J shows that the group treated with acombination of 3 mg/kg anti-PD-1 antibody with 0.5 mg/kg anti-CTLA-4antibody (Group J) had 2 mice that had ulcerated tumors, 2 mice with atumor volume less than 500 mm³, and 4 mice that were tumor free. Theunexpected synergistic effect of an anti-PD-1 antibody combined with ananti-CTLA-4 antibody, along with the surprising effectiveness ofsubtherapeutic levels of anti-CTLA-4 antibody in the combination, areshown in FIGS. 34 (mean tumor volume) and 35 (median tumor volume).

FIG. 36 shows the response to immunostimulatory antibody treatment inmice with larger tumors, those having an initial volume of about 250 mm³(i.e., at the time of the first antibody treatment). FIG. 36A shows thatall 6 control mice (Group K) reached a tumor end-point (4 with a tumorvolume greater man 1500 mm³ and 2 with an ulcerated tumor). FIG. 36Bshows that the group treated with a combination of 10 mg/kg anti-PD-1antibody with 1 mg/kg anti-CTLA-4 antibody (Group L) had one mouse withan ulcerated tumor; 4 mice with a tumor volume greater than 1500 mm³,and one mouse that was tumor free. The mean and median tumor volumes areshown in FIGS. 37 and 38.

The tumor growth inhibition in mice treated with these antibodies, ascompared to mice treated with the control antibody mouse IgG, issummarized in Table 7 and FIG. 39.

TABLE 7 Tumor Growth Inhibition Following Anti- PD-1 and/or Anti-CTLA-4Treatment No. Mean Median Tumor Mice Tumor Tumor Free at Volume -Volume - Mice Tumor mm³ TGI* mm³ TGI (Day End Group (Day 23) (Mean) (Day23) (Median) 51) Point A 700 — 1,380 — — — B 1,710 — 1,360 — — — C 1,05039% 925 32 — — D 770 55% 505 63 — — E 155 91% 100 93 2/8 6/8 F 1,050 39%675 50 — 7/8 G 1,070 37% 1,145 16 — 6/8 H 85 95% 25 98 4/8 3/8 I 75 96%60 95 4/8 1/8 J 80 95% 5 99 4/8 0/8 K 1,900 — 2,125 — — — L 1,115 411,090 49 1/6 — *TGI = tumor growth inhibition; the median could only becalculated when fewer than 50% of the mice reached the tumor end point.^(†) Groups are as defined in FIGS. 33 and 36. For smaller initialtumor: A = vehicle (PBS); B = mouse IgG, 10 mg/kg; C = anti-CTLA-4, 0.25mg/kg; D = anti-CTLA-4, 0.5 mg/kg; E = anti-CTLA-4, 5 mg/kg; F =anti-PD-1, 3 mg/kg; G = anti-PD-1, 10 mg/kg; H = anti-PD-1, 10 mg/kgwith anti-CTLA-4, 0.25 mg/kg; I = anti-PD-1, 10 mg/kg with anti-CTLA-4,0.5 mg/kg; and J = anti-PD-1, 3 mg/kg with anti-CTLA-4, 0.5 mg/kg. Forlarger initial tumor: K = mouse IgG, 10 mg/kg; and L = anti-PD-1, 10mg/kg with anti-CTLA-4, 0.25 mg/kg.

Together these data indicate that the combination therapy comprisinganti-PD-1 and anti-CTLA-4 antibodies is substantially more effectivethan treatment with either antibody alone. In addition, surprisingly thedose of each antibody can be reduced without affecting the synergisticefficacy of this combination of immunostimulatory therapeuticantibodies. The combination therapy still seems to be effective evenwhen the tumor mass is more mature (i.e., larger).

Example 18

Tumor Immunity in Mice Following Anti-PD-1 Antibody Treatment andRe-Challenge with PD-L1⁻ Fibrosarcoma Cells

Mice that survived tumor-free from a challenge with tumor cells andtreatment with anti-PD-1 antibody (i.e., treatment similar to theefficacy studies described in Examples 5 and 6) were then re-challengedwith tumor cells to investigate immunity to tumor formation after such atreatment. Briefly, in the initial challenge, SA1/N fibrosarcoma cells(PD-L1⁻) were implanted subcutaneously in A/J mice (1×10⁶ cells/mouse)on day 0. On days 1, 4, 7, 10, 14, 17 and 20 post-implantation, groupsof mice were injected IP with either mouse IgG (control, 10 mg/kg permouse) or with one of various doses of anti-PD-1 monoclonal antibody 4H2(30, 10, 3, 1 and 0.3 mg/kg per mouse). Tumor formation and volume wasmonitored with a precision electronic caliper twice a week until thestudy was complete. A group of 8 mice were tumor-free after the anti-PD1antibody treatment (4 that were treated with 30 mg/kg, 2 with 3 mg/kg,one with 1 mg/kg, and one with 0.3 mg/kg).

The eight treated, tumor-free A/J mice were re-challenged bysubcutaneously implanting 1×10⁶ SA1/N fibrosarcoma cells/mouse. As acontrol, nine naïve mice were subcutaneously implanted with 1×10⁶ SA1/Nfibrosarcoma cells/mouse. Tumor formation and volume was monitored witha precision electronic caliper twice a week until day 62post-implantation. All nine naïve (control) mice reached the tumorend-point by day 22 post-implantation of the fibrosarcoma cells. Incontrast, the eight tumor-free mice re-challenged with fibrosarcomacells did not develop tumors up to 62 days post-implantation. FIG. 47shows the mean tumor volume for the naïve and re-challenged mice. Theseresults demonstrate that treatment with an immunostimulatory antibody,such as anti-PD-1, provides the treated subject with immunity to furthertumor formation, even in the presence of cells capable of forming atumor.

Example 19 Tumor Immunity in Mice Following Single Antibody Therapy(Anti-PD-1) or Combination Antibody Therapy (Anti-CTLA-4 and Anti-PD-1Re-Challenged with PD-L1⁻Colorectal Cancer Cells

Mice that survived tumor-free from a challenge with tumor cells andtreatment with either anti-PD-1 antibody alone or anti-PD-1 antibodycombined with anti-CTLA-4 antibody (i.e., treatment similar to theefficacy studies described in Examples 2-4) were then re-challenged withtumor cells to investigate immunity to tumor formation after suchtreatments. Briefly, in the initial challenge, MC38 colorectal cancercells (PD-L1⁻) were implanted in C57BL/6 mice (2×10⁶ cells/mouse) on day0. On days 0, 3, 6 and 10 post-implantation, groups of mice wereinjected IP with one of the following treatments: (1) mouse IgG(control, 10 mg/kg per mouse), anti-PD-1 monoclonal antibody 4H2, or (3)anti-PD-1 monoclonal antibody 4H2 in combination with anti-CTLA-4monoclonal antibody 9D9. Tumor growth was monitored with a precisionelectronic caliper as described in Example 15. A group of 11 mice weretumor-free after the anti-PD1 antibody treatment (2 total) or thecombination anti-PD-1/anti-CTLA-4 antibody treatment (9 total).

The 11 treated, tumor-free C57BL/6 mice were re-challenged byimplantation of 2×10⁷ MC38 colorectal cancer cells/mouse (i.e., a doseof cells 10×greater than the initial challenge). As a control, sevennaïve mice were implanted with 2×10⁷ MC38 colorectal cancer cells/mouse.Tumor formation and volume was monitored with a precision electroniccaliper for the duration of the re-challenge experiment (at least 20days). FIG. 48 shows that all seven naïve (control) mice developed atumor and reached the tumor end-point by day 18 post-implantation of thecolorectal cancer cells. In contrast, all 11 tumor-free micere-challenged with colorectal cancer cells did not develop tumors up to18 days post-implantation. FIG. 49 shows the mean tumor volume for thenaïve and re-challenged mice. These data indicate that, similar to theantibody monotherapy, the combination antibody therapy resulting in PD-1and CTLA-4 blockade produces a persistent immunity to tumor relapse.

Example 20 In Vivo Efficacy of Combination Therapy (Anti-CTLA-4 andAnti-PD-1 Antibodies) on Established Tumor Growth

CT26 colorectal cancer cells were implanted in BALB/Cmice (2×10⁶cells/mouse) for a time sufficient (about 10 days) to permit theformation of tumors. On day 10 post-implantation, tumor measurementswere taken and mice were randomized based on mean tumor volume (about250 mm³) into 5 groups for subsequent antibody therapy. At day 0 (i.e.,10 days after the CT26 cells were implanted), mice were injected IP with(1) mouse IgG (control), (2) anti-CTLA-4 monoclonal antibody 9D9, (3)anti-PD-1 monoclonal antibody 4H2, or (4) anti-CTLA-4 monoclonalantibody 9D9 and anti-PD-1 antibody monoclonal antibody 4H2, at aconcentration of 10 mg/kg per mouse. Antibody injections were alsoadministered on days 3, 6 and 10. The monoclonal antibody compositionsused had low levels of endotoxin and did not significantly aggregate.Using an electronic caliper, the tumors were measured threedimensionally (height×width×length) and tumor volume was calculated.Tumor measurements were taken on day 0 (tumors at the beginning oftreatment had a volume of about 125 mm³), and on days 3, 6, 10, 13, 17and 20 post-antibody injection. Mice were euthanized when the tumorsreached a designated tumor end-point (a particular tumor volume such as1500 mm³ and/or when the mice showed greater than about 15% weightloss). The results are shown in FIG. 50. This study indicates that, in amurine tumor model, treatment with the combination of CTLA-4 antibodyand PD-1 antibody has a significantly greater effect on tumor growththan either antibody alone, even when a tumor is already wellestablished.

Example 21 Effect of Human Anti-PD-1 Antibody on Function of TRegulatory Cells

T regulatory cells are lymphocytes that suppress the immune response. Inthis example, T regulatory cells were tested for its inhibitory functionon proliferation and IFN-gamma secretion of CD4°CD25− T cells in thepresence or absence of an anti-PD-1 human monoclonal antibody.

T regulatory cells were purified from PBMC rising a CD4+CD25+ regulatoryT cell isolation kit (Miltenyi Biotec). T regulatory cells were addedinto a mixed lymphocyte reaction (see above) containing purifiedCD4+CD2− T cells and allogeneic dendritic cells in a 2:1 ratio ofCD4+CD25− to T regulatory cells. Anti-PD-1 monoclonal antibody 5C4 wasadded at a concentration of 10 μg/ml. Either no antibody or an isotypecontrol antibody was used as a negative control. Culture supernatantswere harvested on Day 5 for cytokine measurement using a Beadlytecytokine detection system (Upstate). The cells were labeled with³H-thymidine, cultured for another 18 hours, and analyzed for cellproliferation. The results are shown in FIGS. 51A (T cell proliferation)and 51B (IFN-gamma secretion). The addition of anti-PD-1 humanmonoclonal antibody 5C4 partially released inhibition imposed by Tregcells on proliferation and IFN-gamma secretion of CD4+CD25− T cells,indicating that anti-PD-1 antibodies have an effect on T regulatorycells.

Example 22 Effect of Human Anti-PD-1 Antibody on T Cell Activation

In this example, effect of blockade of PD-1 pathway by anti-PD-1antibody 5C4 on T cell activation was examined. Purified human CD4+ Tcells (Dynal CD4 T cell purification kit) were activated with 1 μg/mlsoluble anti-CD3 antibody (BD) in the presence of autologous monocytesor monocyte-derived dendritic cells (DCs). Monocytes were purified usingMiltenyi CD14 monocyte purification kit, and DCs was generated in vitroafter culture of monocytes with GM-CSF and IL-4 (Pepro Tech) for 7 days.After three days of activation in the presence or absence of titratedanti-PD-1 antibody or irrelevant isotype control mAb, culturesupernatants were harvested for ELISA analysis of IFNγ secretion whiletritiated thymidine was added during the final 18 hours of the assay inorder to measure T cell proliferation. The results shown in FIGS. 52Aand 52B demonstrate that PD-1 blockade by anti-PD-1 antibody resulted inenhanced T cell proliferation and IFN-γ secretion. Synergic effect byanti-PD-1 antibody and anti-CTLA-4 antibody on T cell activation(specifically on IFN-γ secretion) in the presence of monocytes was alsoobserved.

Example 23 Assessment of ADCC Activity of Anti-PD-1 Antibody

In this example, an antibody-dependent cellular cytotoxicity (ADCC)assay was performed to evaluate whether anti-PD-1 antibody could induceADCC to target cells. Two versions of 5C4, one with an Fc region ofhuman IgG1 (5C4-IgG1) and the other with an Fc region of human IgG4(5C4-IgG4), were tested in the assay. The Delfia Cell Cytotoxicity Kitfrom Perkin Elmer was used for the assay. Briefly, purified human CD4 Tcells (Dynal CD4 T cell purification kit) were activated by plate-boundanti-CD3 antibody (BD) to induce PD-1 expression. Target activated CD4 Tcells were then labeled with BATDA reagent. Labeled CD4 T cells wereadded to a V-bottom 96-well plate, followed by the addition of humanPBMC (an effector to target (E/T) cell ratio of 50:1) and designedantibody. After incubation for 1 hour at 37° C., the plate was spundown. Supernatant was transferred into a flat bottom 96-well plate andthe plate was read using a RubyStar plate reader. Results showed that5C4-IgG4 did not mediate ADCC on activated CD4 T cells, while 5C4-IgG1did mediate ADCC on activated CD4 T cells (FIG. 53), indicating thatADCC activity is related to its Fc region of the anti-PD-1 antibody.

Example 24 Assessment of Complement-Dependent Cytotoxicity of Anti-PD-1Antibody

In this example, complement dependant cytotoxicity (CDC) of anti-PD-1antibody was examined. Two versions of 5C4, one with Fc region of humanIgG1 (5C4-IgG1) and the other with Fc region of human IgG4 (5C4-IgG4),were tested in the assay. Briefly, purified human CD4 T cells (Dynal CD4T cell purification kit) were activated by plate-bound anti-CD3 antibody(BD) to induce PD-1 expression. Serial dilutions of anti-PD-1 antibody(5C4) and control antibodies from 50 μg/mL to 640 pg/mL were tested forCDC in the presence of human complement (Quidel-A113). Alamar blue(Biosource International) was used to measure cytotoxicity. The platewas read on a flourescent plate reader (EX530 EM590). Viable cell countsare proportional to fluorescence units. Results showed that neither5C4-IgG1 of 5C4-IgG4 mediated CDC on activated CD4 T cells, while thepositive control antibody (anti-HLA-ABC antibody) did (FIG. 54).

Example 25 Assessment of PD-1 Expression on Human T Cells

In this example, human PBMCs from different donors were examined forPD-1 expression on various cell subsets by FACS. Biotinylated anti-PD-1antibody, which has displayed a much higher sensitivity thancommercially available anti-PD-1 antibody on detection of PD-1 moleculeson cell surface, was used in the assay. Bound antibody was detectedusing an PE-conjugated streptavidin. Flow cytometric analyses wereperformed using a FACScan flow cytometry (Becton Dickinson) and Flowjosoftware (Tree Star), PD-1 expression was defected on some peripheralhuman T cells, but not on B cells or monocytes. Further examination of Tcell subsets indicates that PD-1 is expressed on CD4 and CD8 memory andeffector T cells, but absent on naïve CD4 or CD8 T cells.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention, in addition to those described herein, will become apparentto those skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. The invention is, therefore, to be limitedonly by the terms of the appended claims along with the full scope ofequivalents to which the claims are entitled.

1-105. (canceled)
 106. A method of treating a tumor in a subject in needthereof comprising administering a therapeutic dose of a human orhumanized anti-PD1 antibody or an antigen-binding portion thereof to thesubject, wherein an anti-CTLA-4 antibody or an antigen-binding portionthereof has been administered to the subject, prior to theadministration of the anti-PD1 antibody or antigen-binding portionthereof, wherein the administration of the anti-PD-1 antibody orantigen-binding portion thereof treats the tumor in the subject. 107.The method of claim 106, wherein the anti-PD-1 antibody orantigen-binding portion thereof cross-competes for binding to human PD-1with: (a) a reference antibody that comprises a heavy chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 1 anda light chain variable region comprising the amino acid sequence setforth in SEQ ID NO: 8; (b) a reference antibody that comprises a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 2 and a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO: 9; (c) a reference antibody thatcomprises a heavy chain variable region comprising the amino acidsequence set forth in SEQ ID NO: 4 and a light chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO: 11; and (d) areference antibody that comprises a heavy chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO: 6 and a lightchain variable region comprising the amino acid sequence set forth inSEQ ID NO: 13; wherein the anti-PD-1 antibody or antigen-binding portionthereof comprises (i) a heavy chain variable region (V_(H)) and (ii) alight chain variable region (V_(L)) that has at least about 85% sequenceidentity to an amino acid sequence selected from SEQ ID NO: 8, SEQ IDNO: 9, and SEQ ID NO: 11; and wherein the anti-PD-1 antibody orantigen-binding portion thereof binds to human PD-1 with a K_(D) of5×10⁻⁹ M or less.
 108. The method of claim 107, wherein the K_(D) ismeasured by surface plasmon resonance (Biacore) analysis.
 109. Themethod of claim 107, wherein the anti-PD-1 antibody or theantigen-binding portion thereof comprises amino acidsEIVLTQSPATLSLSPGERATLSC (SEQ ID NO:75), WYQQKPGQAPRLLIY (SEQ ID NO: 76)and FGGGTKVEIK (SEQ ID NO: 78).
 110. The method of claim 107, whereinthe VH of the anti-PD-1 antibody or antigen-binding portion thereofcomprises V_(H)-FR1, V_(H)-CDR1, V_(H)-FR2, V_(H)-CDR2, V_(H)-FR3,V_(H)-CDR3, and V_(H)-FR4, and the V_(L) comprises V_(L)-FR1,V_(L)-CDR1, V_(L)-FR2, V_(L)-CDR2, V_(L)-FR3, V_(L)-CDR3, and V_(L)-FR4,and wherein the V_(L)-FR1, V_(L)-FR2, and V_(L)-FR3 comprise amino acidsequences derived from a human V_(K) L6 germline sequence and theV_(L)-FR4 comprises an amino acid sequence derived from a human V_(K)JK1 or V_(K) JK4 germline sequence.
 111. The method of claim 107,wherein the anti-PD-1 antibody is a humanized antibody.
 112. The methodof claim 107, wherein the anti-PD-1 antibody or antigen-binding portionthereof comprises a human constant region of IgG4.
 113. The method ofclaim 112, wherein the human constant region comprises a mutation thatincreases stability of the monoclonal antibody or antigen-bindingportion thereof.
 114. The method of claim 113, wherein the mutation islocated in the hinge region of the constant region.